Method to deliver compositions conferring resistance to alopecia to hair follicles

ABSTRACT

The invention describes a method to deliver a composition selectively to hair follicles using a liposomal formulation. Proteins which are cell cycle inhibitors are products of the multi-drug resistance gene or the recombinant materials for their production are targeted to hair follicles by encapsulating them in liposomes.

This application is a continuation-in-part of pending InternationalApplication No. PCT/US94/03634, filed Apr. 1, 1994, designating theUnited States, which is a continuation-in-part of U.S. application U.S.Ser. No. 08/181,471, filed Jan. 13, 1994, now U.S. Pat. No. 5,641,508,which is a continuation-in-part of U.S. application Ser. No. 08/041,553,filed Apr. 2, 1993, now abandoned, all of which are incorporated byreference (including drawings).

TECHNICAL FIELD OF THE INVENTION

This invention relates to methods for specifically deliveringtherapeutic or other beneficial compounds to hair follicles to improvehair growing from the follicles. These beneficial compounds include, butare not limited to, hair growth stimulators, hair growth inhibitors,compounds used to prevent alopecia, compounds used to restore naturalhair pigment, and compounds used to modify the color of hair.

BACKGROUND OF THE INVENTION

There has been a long-felt need for methods of directly influencing hairgrowth, color and appearance, especially for treatment of alopecia inhumans.

Surgical transplantation of small, discrete, skin areas having viablefollicles to areas having inactive follicles is expensive,labor-intensive and relatively short-lasting. Also, as described by R.F. Oliver et al. in U.S. Pat. No. 4,919,664, follicular dermal cells canbe inserted into a skin incision, resulting in hair growth along theincision. However, this is a complex technique that does nothing tostimulate existing follicles.

Treatment of the hair and skin with various creams or lotions withbiologically active ingredients to improve hair growth and otherconditions has generally low efficiency. A wide variety of externallyapplied agents are available for application to the hair to improvebody, flexibility, curl, etc. These have limited and only short termusefulness. Coloring hair with various dyes requires frequentrepetitions and is not always natural in appearance.

The use of biologically active compounds that are hair growthstimulators or advantageously change other hair characteristics, such ascolor, would seem to be a more natural and attractive approach,especially at the stage where hair-follicle cells still exist but hairgrowth, for unknown reasons, is adversely affected. Attempts to followthis approach have been ineffective, possibly because of the inabilityof stimulators to penetrate the cellular membrane of hair follicle cellsand to enter into the cells where their action is needed.

In the treatment of skin with various absorbable lotions and the like ithas long been known that absorption is generally greater in skin areasof higher follicular density. See, for example, Maigach et al, Arch.Environ. Health, 23:208-211 (1971). The absorbed materials, however,were entirely different from liposomes. It was not appreciated prior tothe present invention that liposomes could be used to direct beneficialcompositions preferentially to hair follicles.

Liposomes, which are artificial phospholipid vesicles, have beensuccessfully used for delivery of different low-molecular-weightwater-soluble and oil-soluble compounds into different cells. See, forexample, G. Gregoriadis, Trends in Biotechnology, 3:235-241 (1985) andK. H. Schmidt, ed., Liposomes as drug carriers, Stuttgart: George ThiemeVerlag (1986).

Liposomes are typically formed by mixing dry phospholipids with aqueoussolutions giving rise to bilayers of phospholipid molecules whicharrange themselves spontaneously to form close multilayered spherules.As they form, the liposomes entrap liquid and any soluble solutes thatare present. A large number of substances that do not interfere with theformation of the liposomes can be incorporated, regardless ofsolubility, electrical charge, size and other structuralcharacteristics. These characteristics may, however, have adverseaffects in some environments limiting the use of liposomes.

Liposomes containing antibody molecules attached for specific targetinghave been described for delivery of encapsulated material to targetedcells containing an antigen immunoreactive with the attached antibody,and are referred to as immunoliposomes. See, for example, U.S. Pat. Nos.4,755,388, 4,925,661 and 4,957,735 for descriptions of immunoliposomes.In addition, liposome compositions have been described that containprotein which are administered to mammalian skin and shown to penetratein skin keratinocytes. See, U.S. Pat. No. 5,190,762. Furthermore,DNA-liposome compositions have also been described, but were not shownto selectively deliver the nucleic acid contents to hair folliclesthrough topical administration. See, U.S. Pat. Nos. 5,077,211 and5,223,263, and Hoffman et al., FEBS Letts., 93:365-368 (1978).

Although various targeting mechanisms have been attempted to increasethe specificity of delivery via liposomes, delivery of the encapsulatedmaterial into a targeted cell or tissue may not necessarily follow.

Specific tissue delivery is particularly important where the agent beingdelivered may have a deleterious effect to tissues adjacent to thetargeted tissue of interest upon administration of the agent. Forexample, the agent may produce effects which are acceptable in the hairfollicle, but not desired in the adjacent skin tissue. For example,delivery of melanin is desirable for hair pigmentation, but may or maynot be desirable for general skin pigmentation, and therefore generaldelivery to all surface skin cells may be undesirable, requiringfollicle cell specificity. Similarly, gene replacement therapy forexpressing melanin or tyrosinase may be undesirable in skin cells, butis a desirable result for hair pigmentation.

Transdermal drug delivery provides additional problems where the drugbeing delivered is destined for the circulation rather than cells of thedermis. Methods for transdermal drug delivery which minimize adsorptioninto cells of the skin and simultaneously increase transport to thecirculation are desirable in certain instances. However, in instanceswhere delivery is directed solely to the hair follicle, it is desirablethat there is minimum adsorption into the skin and minimum transport ofthe compound into the systemic circulation where the administeredcompound can exert undesirable side effects.

A small molecule dye, carboxyfluorescein has been found to be deliveredto the pilosebaceous units of hamster ear membrane when incorporated ina particular liposomal formulation, as described in a very recent paperby Lieb et al, The Journal of Investigative Dermatology, 99:108-113(1992). Similarly, Li et al., In Vitro Cell. Dev. Biol., 28A:679-681(1992), have recently described liposome-mediated delivery of the smallmolecule dye calcein to hair follicles in an in vitro intact skinhistoculture system.

The prior research, however, does not describe methods of specificallyand selectively (preferentially) targeting hair follicles usingliposomes containing large molecule agents such as proteins or nucleicacids, lipophobic agents that cannot transfer across lipid barriers orlipophilic agents which are capable of undesirable side effects ontissues other that hair follicles.

Furthermore, there have not been any descriptions of methods foraccurately testing in vitro the extent to which particular compounds aredelivered to hair follicle cells, the effectiveness of the compoundsdelivered, or liposome formulations for optimizing selectivity oftargeting to hair follicles.

Thus, there is a continuing need for improved methods of selectivelydelivering specific beneficial compounds to hair follicles, and formeasuring effectiveness of the delivery.

SUMMARY OF THE INVENTION

It has now been discovered that liposomes can selectively target thehair follicle with potentially beneficial compounds. The inventiondescribes the unexpected results obtained by the inventors thatliposomes selectively deliver compounds to the hair follicle thusenabling the compounds to cross the stratum corneum and be delivered tothe cells in the hair follicle without delivery of the compounds to thesurrounding skin cells.

The present invention provides compositions and methods which are usefulfor the specific delivery of beneficial compounds to hair follicle cellsto, for example, improve hair color or condition, prevent alopecia, orto stimulate hair growth.

This invention describes methods for preparing liposomes, incorporatingbeneficial compounds into the liposomes either during formation of theliposomes or thereafter, and applying the liposomes to the skin areasrequiring treatment in patients requiring such beneficial treatment.According to the present methods, liposomes preferentially deliver thebeneficial compounds to the hair follicles where the compounds enterinto the follicle cells. By virtue of the selectivity of theliposome-mediated delivery method, the administered compounds are notdelivered substantially to the dermis or internally to the circulation,thereby minimizing undesirable side effects that the administeredcompound might exert on such dermis tissue or systemically in thecirculation.

Thus, the invention provides a method of delivering beneficial compoundsto the hair follicle and thus to the cells in the hair follicle. Theinvention provides a method of delivering any compound to the hairfollicle by the use of liposomes. Although particular liposomecompositions are specifically used as examples herein, the inventionprovides for the use of potentially any liposome to deliver thebeneficial compound. Those of ordinary skill in the art will readilyappreciate that any liposome may be used and that the invention is notlimited to the particular types of liposomes described herein.

Thus, in a first aspect, the invention provides a method of directly andselectively delivering a beneficial compound to hair follicles of amammal comprising the step of applying a liposome composition topicallyto skin areas of a mammal having a plurality of hair follicles, whereinthe liposome composition comprises a liposome containing an effectiveamount of at least one selected beneficial compound, the liposome iscapable of selectively delivering the beneficial compound to the hairfollicle and the beneficial compound is preferentially transmitted tothe hair follicle and enters into the hair follicle withoutsubstantially entering into the cells external to the hair follicle.

By "substantially" is meant that the compound is not appreciablydelivered to the surrounding skin cells. Generally, approximately 5-20%of the compound in the liposome composition that is topically appliedenters into the cells of the treated skin tissue. More commonly, 10% ofthe liposome composition topically applied enters the cells of thetreated skin tissue. By "substantially" is meant that not more than 10%of the compound that enters the cells is delivered to the cells externalto the hair follicle. More preferably, not more than 1% of the compoundthat enters the cells is delivered to the cells external to the hairfollicle cells. In especially preferred embodiments, not more than0.1-0.5%, preferably 0.1%, of the compound that enters the cells isdelivered to cells external to the hair follicle.

Thus, one advantage of the invention is that beneficial compounds may bedirectly and selectively delivered to cells in the hair follicle withoutentering into other cells, such as other cells in the skin or the bloodstream.

By "directly and selectively" is meant that the invention delivers thecompound preferentially to the hair follicle and does not substantiallydeliver the compound to the cells surrounding the hair follicle that arenot hair follicle cells or to the systemic circulatory system.

The present invention thus yields the surprising result that beneficialcompositions may be directly and selectively delivered to hair follicleswithout transmission to the cells external to the hair follicles. Themechanism by which this selective transmission may take place may be,for example, but not by way of limitation, due to an attraction of theliposome compositions to the oil secreted by the sebaceous glandsassociated with the hair follicles.

Typically, the present methods are practiced on the skin of a mammalrequiring treatment according to the present methods, such as a human.Thus, the methods can be practiced in vivo.

In order to determine the effectiveness of the hair follicle-specifictreatment method of the present invention, an in vitro method of testingparticular liposome agents has been developed, utilizing novelhistoculturing techniques.

As mentioned above, it is known that a number of compounds, typicallydyes and the like, when applied to the skin are more rapidly absorbed inheavily follicularized areas. However, many macromolecular or lipophobicsubstances cannot cross the plasma membrane or other lipid barriers intothe follicle and follicle cells. In the present invention, it has beendiscovered that when incorporated into liposomes, those macromolecularcompounds are successfully transported into the follicle cells, andfurthermore can be selectively transferred across the stratum corneuminto the follicle without entry to the circulation or the adjacent skintissue, which has great potential efficacy as well as safety advantages.

Thus, the invention describes in one embodiment a liposome compositioncomprising a liposome containing an effective amount of a beneficialcompound. The liposome utilized in the liposome composition is capableof selectively delivering the beneficial compound to hair follicles asdescribed further herein. The beneficial compound to be administered canbe a macromolecule or lipophobic molecule that is not capable of passagethrough the stratum corneum or cell membrane and requires theliposome-mediated delivery system to selectively and preferentiallyenter the hair follicle, or is a lipophilic molecule having undesirableeffects on cells external to the hair follicles, and requires theselectivity of the liposome-mediated delivery system to preferentiallydeliver the lipophilic molecule to the hair follicle.

The liposome compositions can be utilized for a variety of applications,as described herein, and therefore may contain any of a variety ofbeneficial compounds, including hair color-restoring or modifying agentssuch as melanin, hair dye, tyrosinase, or a nucleic acid which iscapable of expressing human tyrosinase, hair growth stimulating or hairfortifying agents, agents which inhibit sensitivity tochemo-therapeutics, other agents which prevent all forms of alopecia andthe like beneficial compounds. These beneficial compounds may thereforebe, for example, but are not limited to, proteins, peptides, nucleicacids, polymers, macromolecules, or dyes.

A liposome composition can comprise any of a variety of liposomesdesigned to selectively target hair follicles, including pH-sensitiveliposomes, liposomes comprising a phospholipid selected from the groupconsisting of phosphatidylcholine (PC), egg phosphatidylcholine (EPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidyl choline(DPPC), phosphatidylethanolamine (PE), dioleoylphosphatidylethanolamine(DOPE) and cholesterol, liposomes further comprising a cationicphospholipid selected from the group consisting of D282, D378, D383,D3886, D3897 and D3899, (obtainable from Molecular Probes Catalog,Eugene, Oreg.) and the like formulations.

The invention also describes a method for restoring hair color to thehair of a mammal, comprising applying a therapeutically effective amountof a liposome composition to a skin area on said mammal having aplurality of hair follicles, where the liposome composition of thepresent invention comprises a liposome containing an effective amount ofat least one selected hair color-restoring agent. Preferred haircolor-restoring agents include melanin, hair dye, tyrosinase, and anucleic acid capable of expressing human tyrosinase in hair folliclecells.

The invention further describes a method of directly and selectivelydelivering a beneficial compound to hair follicles of a mammalcomprising the step of applying a liposome composition of this inventiontopically to skin areas of a mammal having a plurality of hairfollicles, wherein the liposome composition comprises a liposomecontaining an effective amount of at least one selected beneficialcompound and wherein the beneficial compound is a macromolecule, alipophobic molecule or a lipophilic molecule having undesirable effectson cells external to said hair follicles. The liposome composition maybe applied to the skin area of a mammal having a plurality of hairfollicles either in vivo, or in vitro, using explanted skin tissue. Theexplanted skin tissue may be grown, for example, as described herein, inskin histoculture. In preferred embodiments, the beneficial compound isa hair color-restoring agent such as melanin, hair dye, or tyrosinase.In related embodiments, the beneficial compound is a hair growthstimulator such as cyclosporin-A, or related compounds, finesteride, oran antisense nucleic acid molecule that would block a gene conferring anegative effect to the hair. Techniques of designing antisense moleculesare well known to those of ordinary skill in the art. Hair growthstimulating compounds may have undesirable side effects when deliveredsystemically, one advantage of the present invention provides compoundsfor and a method of directly and selectively delivering the compounds tothe hair follicle cells without substantially delivering the compound tothe bloodstream, thus avoiding such undesirable side effects. In anotherrelated embodiment, the beneficial compound is a nucleic acid capable ofexpressing an effective amount of a replacement therapy protein.Particularly preferred are nucleic acid molecules capable of expressingtyrosinase or hair-growth stimulating proteins or the multi-drugresistance proteins conferring resistance to chemotherapy-inducedalopecia.

In other embodiments, the invention contemplates the use of the presentliposome compositions according to the present methods for inhibitingchemotherapy-induced alopecia. The liposome compositions containcompounds which reduce in the hair follicle the toxicity of thechemotherapy treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

Details of the invention, and of certain preferred embodiments thereof,will be further understood upon reference to the drawings, wherein:

FIG. 1A is a fluorescent microscopy image (magnification 500×) of a skinhistoculture treated with liposomes containing calcein as described inExample 1 showing highly preferential dye delivery into hair follicles;

FIG. 1B is a fluorescent microscopy image (magnification 500×) of a skinhistoculture treated with calcein without liposomes as described inExample 1, showing weak dye staining and no preferential delivery toskin structures;

FIG. 2 is a hematoxylin and eosin stained paraffin-section ofwhite-haired mouse skin treated with melanin entrapped liposomes for 12hours, (magnification 500×) as described in Example 2, showing that theliposome-entrapped melanin primarily delivered the melanin to hairfollicles as indicated by the arrows;

FIG. 3 is a light microscopy image (magnification 250×) of a paraffinsection of skin histoculture as in FIG. 2 stained with hematoxylin andeosin as described in Example 2, showing delivery of melanin into thehair shaft itself as indicated by the arrows;

FIG. 4 is a histological autoradiogram of histocultured skin treatedwith liposomes entrapped with radioactive labeled high-molecular weightDNA showing the localization of DNA (arrows) in hair follicle cellmembrane and cytoplasm, as described in Example 3;

FIGS. 5A-5D contain light microscopy images (magnification 125×, FIGS.5A and 5C; magnification 250×, FIGS. 5B and 5D) of sections of skinhistoculture prepared as described in Example 4b, in which FIGS. 5A and5B illustrate results using liposome-entrapped plasmid(pM-MuLV-SV-Lac-Z) capable of expressing Lac-Z, and FIGS. 5C and 5Dillustrate results using naked plasmid. The arrows indicate uniformdistributions of blue (dark) spots in the hair follicles and shaftsindicating active gene transfer to the hair follicles;

FIGS. 6A-6C contain fluorescent light microscopy images (magnification150×) of sections of mouse skin samples prepared by treatment of mouseskin in vivo as described in Example 5, in which FIGS. 6A and 6Billustrate results using liposome-entrapped calcein and FIG. 6Cillustrates results using naked calcein. The arrows indicatefluorescence in the hair shafts indicating active transfer of calcein tothe hair follicles;

FIGS. 7A-7C contain light microscopy images (magnification 500×) ofsections of mouse skin samples prepared by treatment of mouse skin invivo as described in Example 5, in which FIGS. 7A-7C illustrate resultsusing liposome-entrapped melanin; and

FIGS. 8A-8F shows the chemical structure of the cationic phospholipidsD282, D378, D383, D3886, D3897 and D3899, respectively.

FIGS. 9A and 9B contain light microscopy images of propidiumiodide-stained skin histoculture treated with calcein entrapped inliposomes (9A) and free calcein (9B) as described in Example 1.

FIGS. 10A and 10B contain light microscopy images of skin histoculturetreated with calcein entrapped in liposomes (10A) and free calcein (10B)as described in Example 1.

FIGS. 11A-C contain images of propidium iodide histocultured skintreated with melanin entrapped in liposomes. FIG. 11A is a lightmicroscopy image and FIGS. 11B and 11C are histological sections of thesame sample as FIG. 11A, as described in Example 2.

FIGS. 12A and 12B contain light microscopy images of mouse skin treatedwith melanin entrapped in liposomes (12A), and mouse skin treated withnaked melanin (12B), as described in Example 2.

FIGS. 13A and 13B contain photographs of mice treated with melaninentrapped in liposomes (13A) and with naked melanin (13B), as describedin Example 2.

FIGS. 14 A-E are light microscopic images of β-galactosidase expressionin mouse hair follicle cells after topical application ofliposome-entrapped Lac-Z. Expression of β-galactosidase results in bluestaining of X-gal substrate in hair follicle bulbs (14A-C) and in thebulge area below the opening of the sebaceous gland (14 D&E) asdescribed in Example 4. Magnification 200×(A), 400×(B), 1000× (c,d,e).

The drawings are not necessarily to scale, and certain features of theinvention may be exaggerated in scale and shown in schematic form in theinterest of clarity and conciseness.

DETAILED DESCRIPTION OF THE INVENTION A. Liposome-Mediated TargetedDelivery of Macromolecules and Nucleic Acids to Hair Follicles

The invention relates to the administration of active compositionsdirectly and selectively (specifically) to the cells of the hairfollicle and to the hair shaft itself.

Because of the hair follicle specificity for delivery according to thepresent invention, the present invention provides the advantage ofspecifically delivering beneficial compositions to the hair folliclerather than generally to the dermis or circulation, thereby allowing theuse of lower amounts of the composition to achieve the desired effect,and thereby reducing the likelihood of undesirable effects caused by thecomposition on the skin generally or to the general circulation.

1. Skin Histoculture Assay

In order to demonstrate that liposomes encapsulating beneficialcompounds are effective at selective delivery, and to provide a meansfor optimizing liposome mediated delivery formulations, an in vitroassay has been developed. Basically, pieces of skin containing hairfollicles are histocultured on collagen-gel-supported sponges asdescribed by Li et al, Proc. Natl. Acad. Sci. USA, 88:1908-1912 1991);Li et al, Proc. Natl. Acad. Sci. USA 89:8764-8768, 1992; Li et al., InVitro Cell. Dev. Biol. 28A:479-481, 1992; Li et al., In Vitro Cell. Dev.Biol. 28A:679-681, 1992; Li et al., In Vitro Cell. Dev. Biol.28A:695-698, 1992; Li et al., In Vitro Cell. Dev. Biol. 29A:192-194,1993; and Li et al., In Vitro Cell. Dev. Biol. 29A:449-450, 1993, theteachings of which are hereby incorporated by reference. The systemallows the growth of hair shafts in the follicle cells for periods of atleast 10-16 days, and further allows the ability to evaluate thethree-dimensional appearance of the hair follicle and surrounding tissueby the use of selective dyes and stains in confocal microscopy, therebyproviding a system for evaluating the effectiveness of the therapeuticreagent being applied The use of the three-dimensional histoculture inconjunction with confocal microscopy allows the ability to follow thefine details of candidate beneficial (therapeutic) product-deliveringliposome interactions with hair-follicles at the cellular andsubcellular level. Therefore, the histoculture system allows the abilityto optimize liposome compositions as well as determine the optimumconditions for delivery of the liposome contents into the target cell.

Typical skin histoculture preparation methods are also detailed incopending U.S. patent application of Li et al., Ser. No. 08/129,022,filed Sep. 29, 1993, and assigned to the assignee of this application,which is a continuation of Ser. No. 07/662,239, filed Feb. 28, 1992,abandoned, and assigned to the assignee of this application, and incopending International Patent Application Serial No. PCT/US92/01571,filed Feb. 28, 1992, and published on Sep. 17, 1992 as InternationalPublication No. W092/15700 the teachings of which are all herebyincorporated by reference.

Native-state histoculturing of a skin sample having hair follicles andinternal and external surfaces comprises placing the skin sample on anextracellular support matrix immersed in a medium whereby the internalsurface is adjacent to the matrix and the external surface is exposed inthe air above the surface of the medium and maintaining the matrix withthe skin thereon under skin culturing conditions.

Potentially any skin from any animal can be used in this assay.Preferably, the animal is a mammal. Exemplary mammals are mice, rats,guinea pigs, hamsters, rabbits, marmosets, monkeys and humans. Morepreferably, the animal is a human.

The skin sample having dermal and epidermal layers is typically excisedfrom an animal. Excess fat, if present, is removed. The sample of skinmay be excised from a hairy animal whose skin is capable of supportinghair growth or from a hairless animal whose skin is devoid of hair, suchas an athymic, nude animal. Where the skin sample is obtained from ahairy animal, the skin may be shaved or clipped prior to excision.

The skin sample is defined herein as having internal and externalsurfaces. The phrase "internal surface" refers to the dermally-orientedsurface; i.e. the non-exposed surface of the skin as it exists in itsnative-state in the animal. The phrase "external surface" refers to theepidermally-oriented surface: i.e. the exposed surface of the skin as itexists in its native-state in the animal.

There is no real limitation as to the surface area of a piece of skinused in the present invention. Typically, the skin sample can range inexternal surface area from about 1 to about 10,000 square millimeters(mm²). A preferred surface area is from about 4 to about 100 mm². A morepreferred surface area is about 10 mm². The thickness of the skin is afunction of the animal from which it is obtained. Where the skin sampleis excised from a mouse, a preferred thickness is about 1 to 2 mm.

Skin samples are cultured on a support matrix. A support matrix of thisinvention provides a trabecular structure with interstices suited forcapillary action to deliver aqueous nutrients from the medium to theinternal surface (base) the skin as in a native state. Thus, any supporthaving this capacity is contemplated including synthetic meshes such asnylon, borosilicate glass fiber, or polypropylene or organic meshes suchas cellulose or collagen. Preferably, the support matrix is anextracellular support matrix. As used herein, the phrase "extracellularsupport matrix" means a solid, such as a gel or sponge, comprising oneor more organic molecules or molecular aggregates, which molecules oraggregates are those produced and secreted by cells into theextracellular space and which serve, in vivo, as a support, adhesive andframework for maintaining three-dimensional tissue organization andfunction. Exemplary of such molecules are high-molecular weight proteinsand glycoproteins such as collagen, laminin, fibronectin and the like,complex polysaccharides and the like molecules.

In a preferred embodiment, the extracellular support matrix is acollagen-containing gel. Exemplary collagen-containing gels aregelatinized pig skin such as GELFOAM™ (The Upjohn Company, Kalamazoo,Mich.) and a composition comprising laminin, collagen, proteoglycan andentactin such as MATRIGEL™ (Collaborative Research, Inc., Bedford,Mass.). GELFOAM™ is a patented product described in U.S. Pat. No.2,465,357, the disclosure of which is incorporated herein by reference.

In another preferred embodiment, the extracellular support matrix is ahomopolysaccharide sponge (Leighton, J., J. Nat'l Cancer Instit.12:545-561, 1951). A preferred homopolysaccharide is cellulose.Homopolysaccharide sponges contemplated by the present invention are notlimited as to weave or net size.

In still another preferred embodiment, the extracellular support matrixcomprises a combination of a collagen-containing gel and ahomopolysaccharide sponge. Preferably, such a combination comprises atop layer of a collagen-containing gel and a bottom layer of ahomopolysaccharide sponge. The collagen-containing gel is preferablygelatinized pig skin and the homopolysaccharide is preferably cellulose.In a particularly preferred embodiment, the support matrix comprises acombination of a top layer of GELFOAM™ and a bottom layer of a cellulosesponge, which matrix has been shown to be most effective in maintainingnormal hair growth of histocultured skin.

There are no set ratios of skin sample size to size of the extracellularsupport matrix. The matrix can be anywhere from a diameter which issufficient to support the skin sample to being greater in size andsubstantially overlapping the skin sample. Multiple samples can beplaced on the same matrix so long as the skin samples are not actuallytouching. A preferred distance between skin samples is about 1 to 2 mm.

The skin sample is placed on the matrix such that the internal surfaceof the skin is adjacent to the matrix and the external surface of theskin is facing away from the matrix. In a preferred embodiment, theinternal surface of the skin is in contact with the matrix. In thisarrangement, the external surface of the skin is available forcontacting with toxins or other compositions to assess their effect onthe skin according to the present methods.

The matrix with the skin sample thereon is immersed in a volume of amedium sufficient to contact the matrix but not to completely cover theskin; i.e. the external surface of the skin is not submerged but isexposed above the surface of the medium. Preferably, the surface of themedium is within 0.5 to 2 mm of the upper surface of the matrix andprovides aqueous contact to the skin sample through a wicking effect.For example, where the skin sample has a thickness of about 1 to 2 mm,the surface of the medium is preferably from about 0.5 to about 2millimeters below the external surface of the skin.

The extracellular support matrix is typically soft and may indent uponplacement of the skin sample thereon such that the edges of the matrixmay contact the vertical edges of the skin sample.

The extracellular support matrix is pre-treated to equilibrate thematrix with the medium before the skin sample is placed thereon.Pretreatment of the matrix comprises cutting the matrix to apredetermined size and soaking the cut matrix in the medium in a sterilecontainer for a period of time sufficient to saturate and equilibratethe matrix with the medium. A preferred soaking time is 4 hours at 37°C.

The medium contemplated by the present invention is an aqueous nutrientmedium designed to promote and maintain viability of the skin sample. Apreferred medium is Eagles Minimum Essential Medium (MEM) supplementedwith 10% (v/v) fetal bovine serum (FBS) and an antibiotic. Exemplaryantibiotics are gentamicin, streptomycin, penicillin, kanomycin and thelike. A preferred antibiotic is gentamicin. The final concentration ofantibiotic in the medium depends upon the particular antibiotic used.Where the antibiotic is gentamicin, a preferred concentration is about0.2 mgs per ml of medium. Other media can also be used, preferablyinvolving the use of fetal bovine serum, or using serum-free definedmedia as is well known in the art.

The matrix with the skin sample thereon may be maintained in the mediumfor indefinite periods of time. Preferably, the medium is changed every2 to 3 days.

After a suitable histoculturing period, a quantity of liposomescontaining the selected beneficial macromolecular compound is applied tothe skin histoculture. A second histocultured skin sample is treatedwith the compound alone as a control. The skin histocultures are thenprocessed and prepared to asses the viability of the tissues and theskin cell undergoing the treatment, and to determine the specificity ofdelivery of the beneficial compound in the liposomes.

In one embodiment, viability and/or delivery is assessed by measuringthe incorporation into cells of the skin sample of an indicator specificfor viable cells. As used herein, the phrase "specific for viable cells"means that the indicator is taken up or incorporated into living, butnot dead, cells.

The indicator specific for viable cells may be a metabolic precursor ora non-metabolite that gains access to living cells. Exemplary metabolicprecursors are ribo- or deoxyribonucleic acid precursors such aspurines, pyrimidines, nucleosides and nucleotides. Preferably, themetabolic precursor is operatively linked to an indicating means tofacilitate detection. A preferred indicating means for ametabolic-precursor indicator is a radiolabel such as ³⁵ S, ³² P, ¹²⁵ I,³ H and the like. A particularly preferred radiolabeledmetabolic-precursor indicator is ³ H-thymidine.

A preferred non-metabolite indicator specific for viable cells is a dyethat is capable of optical detection. Any dye recognized in the art asbeing specific for viable cells can be used in accordance with the skintoxicity assay of this invention. See, e.g., Handbook of FluorescentProbes and Research Chemicals, ed. by R. P. Haugland, Molecular Probes,publisher, Eugene, Oreg. (1989-1991 and 1992-1993).

In a preferred embodiment, the dye is a fluorescent dye. Exemplaryviable-cell-specific fluorescent dyes are BCECF-AM (B-1150), Calcein-AM(C-1430), CFDA (carboxyfluorescein diacetate; C-195) Acridine orange(A-1301), Calcein blue (H-1426), Fura-2AM (F-1201), Fluoresceindiacetate (F-1303) or Carboxy analog (C-1431) and the like. Such dyesare well known in the art and are commercially available (MolecularProbes, Eugene, Oreg.). Particularly preferred are the dyes BCECF-AM orCalcein-AM. The numerals in the parenthesis indicates the product numberfor the listed fluorescent dyes that are available from MolecularProbes.

In one embodiment, the incorporation or uptake of fluorescent dyesspecific for viable cells depends upon metabolic activity of the viablecell. In accordance with this embodiment, non-fluorescing dyes are takenup by viable cells and converted to a fluorescing product by anintracellular enzyme such as an esterase. The presence of intracellularfluorescence indicates viability.

In another embodiment, viability is assessed by measuring the uptake orincorporation into cells of the skin sample of an indicator specific fordead cells. As used herein, the phrase "specific for dead cells" meansthat the indicator is taken up or incorporated only into dead,non-viable cells.

Typically, dyes specific for dead cells are compounds with a high ioniccharge and low permeability such that the dyes cannot permeate intactcellular membranes. When cells die, the membrane is structurally orfunctionally ruptured such that dyes specific for dead cells gain accessto the intracellular space where they bind to intracellular componentssuch as nuclear membranes.

A preferred dead-cell-specific indicator is a dye capable of opticaldetection. A preferred dead-cell-specific dye is a fluorescent dye suchas propidium iodide, ethidium bromide, ethidium homodimer(5,5'-diazadecamethylene) bis (3,8-diamino-6-phenylphenanthridium)dichloride, dihydrochloride! and the like. most preferred is propidiumiodide. Propidium iodide (PI) and other dyes specific for dead cells arewell known in the art and commercially available (Molecular Probes,Eugene, Oreg.).

In still another preferred embodiment, assessing viability isaccomplished by simultaneously measuring the uptake or incorporation ofboth an indicator specific for viable cells and an indicator specificfor dead cells. Viability is assessed as the ratio of viable to deadcells. Where both the indicator specific for viable cells and theindicator specific for dead cells are fluorescent dyes, such dyes shouldhave different emission spectra so as to facilitate discriminationbetween viable and dead cells. Compositions and methods for determiningcell viability by the differential uptake of indicators specific forviable and dead cells and tissue culture samples are well known in theart. Haugland, Supra.

Means for detecting the uptake or incorporation of indicators specificfor viable cells are dependent upon the particular indicator used andare well known to those of skill in the art. A preferred means fordetecting radiolabeled metabolic-precursors is autoradiography ofhistological sections of the skin samples that have taken up theprecursor.

A preferred means for detecting dyes is microscopic examination.Microscopic examination can involve the use of any microscope thatallows one to selectively and reproducible evaluate indicatorincorporation into specific cells of the skin sample at varyinglocations within the three-dimensional, native-state skin histoculture.

Typically, the microscopic examination requires the capability ofoptical sectioning. Optical sectioning is the ability to viewpreselected depths within the three-dimensional structure of the skin inthe absence of optical interference provided by the presence in the skinof microsomes, air bubbles, fat globules and other tissue components,which provide reflection of light and optical interference.

In addition, optical sectioning allows for viewing a variety of planeswithin the three-dimensional skin histoculture. By sequentiallysectioning serial layers of the skin, one can produce a total picture ofthe skin and hair follicle or, alternatively, a picture of a region ofthe skin and the follicles where a particular cell type of interest islocated. Thus, comparative studies of a plurality of depths or regionsof the skin can be made. In this way, viability can be assessed insurface cells, at cells underneath the dermal layer, cells inside theepidermal layer, or in other specific cell types such as nerve cells,oil secreting cells, hair follicle cells.

The optical section thickness can be varied to accommodate the cell sizeor tissue to be observed and can range from about 0.1 to 1000 microns.Preferred sections are in the range of 0.5 to 10 microns, preferablyabout 2 to 6 microns.

A preferred microscope that is capable of performing optical sectioningis a confocal scanning laser microscope such as the MRC-600 CONFOCALIMAGING SYSTEM (Bio-Rad, Richmond, Calif.), mounted on a Nikon Optiphotusing a 10× PlanApo plan objective. Such a confocal scanning microscopehas been successfully used to asses delivery (see the Examples). Otheravailable methods for optically scanning or sectioning planes of thetissue sample are also contemplated by the present invention.

Viability is assessed at any particular location within the skin as aratio of viable or dead cells to total cells or as a ratio of live todead cells on the basis of the uptake of indicators specific for viableand dead cells respectively. When viability is assessed both before andafter contact with a putative beneficial compound, comparing the ratioof live to dead cells as assessed before and after contact with theputative beneficial agent provides an indication of the toxicity orbenefit provided by the administered compound.

The procedure for applying indicators to the skin culture varies withthe particular indicator used. Typically, indicators are added to themedium about 6 hours and, preferably about 24 hours after placing theskin sample in the medium. Following addition of the indicator to themedium, the culture is maintained under culturing conditions for aperiod time sufficient to allow the indicator to enter and label thecells of the skin sample. Preferably, the culture is maintained in thepresence of the indicator for about 5 minutes to about 2 hours and, morepreferably for about 10 to 20 minutes.

The concentration of indicator added to the medium varies with theparticular indicator used. Where the fluorescent dyes PI and BCECF-AMare used, the dye concentration is from about 1 to about 100 micromolar,preferably from about 2 to about 50 micromolar, and more preferablyabout 5 micromolar each.

Exemplary in vitro skin histoculture methods are described in theExamples.

The in vitro histoculture assay can be utilized in a variety of ways.The assay can be utilized to evaluate and optimize liposome formulationsfor enhanced efficacy of delivery of the beneficial compound, or tostudy other aspects of the liposomes usefulness in the targetingformulation. Furthermore, the assay can be used as a screening system toidentify additional beneficial compounds for treating conditionsafflicting hair follicles as described further herein.

In addition, the in vitro histoculture assay methods can be utilized todetermine the effective dosages of beneficial compounds for use in thepresent methods.

2. Preparation of Liposomes Encapsulating Beneficial Compounds, andLiposome Compositions

A beneficial liposome composition of the invention is typically providedin one or more of a variety of compositional forms suitable for thecontemplated use. Although proteins, nucleic acids or other compoundsfor use in a liposome generally retain biological activity in a varietyof buffers and solutions, it is preferred to be formulated in aphospholipid composition. Particularly preferred are phospholipidcompositions which afford maximum stability and biological activity ofthe beneficial compound in the composition. Such phospholipidcompositions are preferably formulated to form liposome compositions, asare generally well known in the art. Typically, the composition containsan amount of biologically active beneficial compound suitable for itscontemplated use.

The preparation of liposomes, and their use in drug therapy has beenpreviously described. See, for example, U.S. Pat. Nos. 4,241,046,4,394,448, 4,529,561, 4,755,388, 4,828,837, 4,925,661, 4,954,345,4,957,735, 5,043,164, 5,064,655, 5,077,211 and 5,264,618, thedisclosures of which are hereby incorporated by reference. Exemplarymethods for the entrapment of nucleic acids into liposomes are describedby Hoffman et al., FEBS Ltrs 93:365-68, 1978 and in U.S. Pat. No.5,223,263, hereby incorporated by reference herein.

Preferred and exemplary methods for preparing beneficialcompound-encapsulated liposomes for use in the present methods aredescribed in the Examples. In particular, the encapsulation of melanin,protein or nucleic acid, each for delivery to hair follicles as abeneficial compound, are described herein.

The liposome compositions of the present invention typically compriseabout 0.1 mg to about 3 mg of protein, or about 0.1 ug to about 0.5 mgnucleic acid, per mg of phospholipid mixture.

The ratio of active compound to phospholipid mixture may determine thesensitivity of the resulting reagent. Thus, use of a ratio of about 1 to2 mg protein per mg phospholipid mixture may be suitable for a proteinreagent having a International Sensitivity Index ("ISI") of about 1.0.Use of a ratio of about 0.25 to about 0.5 mg protein per mg phospholipidmixture may be suitable to prepare a composition having an ISI of about1.6 to about 2.0.

Preferred are compositions that additionally comprise from about 0.5 toabout 1.5% (w/v) glycine. Where it is desired to be able to lyophilizethe liposome composition to allow storage and later reconstitution, thereagent preferably includes a cryopreservative, preferably acarbohydrate preservative, most preferably trehalose.

The lipid bilayer of the liposomes comprises phospholipids, preferably,phosphoglycerides. Exemplary liposome compositions includephosphatidylcholine (PC) liposomes, particularly egg PC (EPC) anddipalmitoyl PC (DPPC). Additional candidate liposome compositions areprepared according to the teachings of U.S. Pat. No. 4,394,488, theteachings of which are incorporated by reference, particularly thedescriptions of liposomes comprising phosphotidylethanolamine (PE),phosphotidylserine (PS), sphingolipids, phosphotidylglycerol (PG),phosphatidic acid (PA), cholesterol, spingomyelin cardiolipin, variouscationicphospholipids glycolipids, gangliosides, cerebrosides and thelike, used either singularly or in combination.

"Phospholipid" refers to an organic molecule derived from eitherglycerol (most commonly) or sphingosine. Phospholipids derived fromglycerol (or phosphoglycerides) comprise a glycerol backbone, two fattyacid chains esterified to the first and second carbons of the glyceroland phosphoric acid esterified to the third carbon. Optionally, analcohol moiety is esterified to the phosphoric acid.

Suitable phospholipids for use in the liposome compositions of thepresent invention include those which contain fatty acids having twelveto twenty carbon atoms; said fatty acids may be either saturated orunsaturated. The phospholipids may come from any natural source and thephospholipids, as such, may be comprised of molecules with differingfatty acids. Phospholipid mixtures comprising phospholipids fromdifferent sources may be used. For example, PC, PG and PE may beobtained from egg yolk; PS may be obtained from animal brain or spinalchord. These phospholipids may come from synthetic sources as well.

Phospholipid (PL) mixtures having a varied ratio of individual PLs maybe used. However, although the phospholipids may be used in variedratios, mixtures of phospholipids having preselected amounts ofindividual phospholipids result in liposome compositions havingadvantageous activity and stability of activity. Thus although a widerange of ratios of individual phospholipids may be used, foradvantageous activity and stability of the resulting liposomecomposition, certain phospholipid compositions are preferred.

The phospholipids are conveniently combined in the appropriate ratios toprovide the PL mixture for use in preparing the liposome composition ofthe present invention.

Liposomes are preferably prepared using one or more phospolipidsincluding (N-(1-(2,3-dioleolyoxy)propyl)-N,N,N-trimethyl ammoniumchloride) (DOTMA), dioleoylphosphatidylethanolamine (DOPE),dioleoylphosphatidylcholine (DOPC), phosphatidylethanolamine (PE), eggPC (EPC), phosphatidylcholine (PC), dipalmitoyl PC (DPPC), cholesteroland the like phospholipids. Phospholipids can be obtained from a varietyof sources, including Avanti (Birmingham, Ala.), GIBCO BRL(Gaithersburg, Md.) and Aldrich (Milwaulkee, Wis.), or can be preparedfrom available materials, as is well known.

Preferred liposomes comprise PC, EPC, or DPPC homogeneously. Furtherpreferred liposome compositions comprise a combination of a PC-typephospholipid (such as PC, EPC, DOPC, DPPC and the like) combined with aPE-type phospholipid (PE, DOPE and the like) in a molar ratio of fromabout 2:5 to about 5:2, more preferably about 5:2 PC:PE. A preferredliposome composition comprises PC:PE:Chol in a molar ratio of 5:2:3.

A preferred liposome for use in the present invention additionallyincludes cationic phospholipids. One preferred cationic phospholipid isa monocationic phospholipid having two identical alkyl side chains.

Preferred cationic phospholipids are also generally available from avariety of sources, including the above recited sources. Particularlypreferred cationic phospholipids include cationic phospholipids such asD282, D378, D383, D3886, D3897 and D3899, obtainable from MolecularProbes (Eugene, Oreg.), the structure and synthesis of which is wellknown and described in Handbook of Fluorescent Probes and ResearchChemicals, ed. by R. P. Haugland, Molecular Probes, publisher, Eugene,Oreg. (1989-1991, and 1992-1993). The structures of cationicphospholipids D282, D378, D383, D3886, D3897 and D3899 are shown in FIG.8.

D282 is also known as1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; D378is also known as 3,3'-diheptyloxacarbocyanine iodide; D383 is also knownas 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate;D3886 is also known as1,1'-dioleyl-3,3,3',3'-tetramethylindocarbocyanine methanesulfonate;D3897 is also known as N-4-(4-dilinoleylaminostyryl)-N-methylpyridiniumiodide; and D3899 is also known as1,1-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate.

In one embodiment, the liposome composition of this invention containsone or more of the above cationic phospholipids. Preferably, a liposomecomposition of this invention comprises a formulation of phospholipidscomprising a mixture of (a) one or more of the phospholipids PC, EPC,DOPC, DPPC, PE, DOPE, cholesterol and the like phospholipids, and (b)one or more of the cationic phospholipids D282, D378, D383, D3886,D3897, D3899 and the like. A particularly preferred liposome compositioncomprises a mixture of phospholipid (a) and cationic phospholipid (b) ina ratio of about 0.5 to 2.0 moles of phospholipid (a) to about 0.5 to1.5 moles of phospholipid (b), and more preferably about 1.0-1.2 molesof phospholipid (a) to 0.8 moles of cationic phospholipid (b). Apreferred phospholipid composition in this embodiment comprises amixture of DOPC or DOPE with one or more of the above cationicphospholipids in a ratio of about 0.8 moles to about 1.0-1.2 moles.

In another embodiment, the invention comprises a liposome compositioncomprising one or more phospholipids selected from the group consistingof PC, EPC, DOPC, DPPC, PE, DOPE and cholesterol, combined with one ormore phospholipids to form pH-sensitive liposomes. pH-sensitiveliposomes are generally well known and their preparation has beendescribed by Straubinger et al., FEBS Letts. 179:148-154, 1985. Apreferred pH sensitive liposome comprises oleic acid (OA) and PE at amolar ratio of 3:7. OA is available from a variety of commercialsources, including Sigma (St. Louis, Mo.). Several pH-sensitive liposomesystems have been described. There are two main categories:intrinsically pH-sensitive liposomes and those which utilize an externalnon-lipid trigger. Intrinsically pH-sensitive liposomes are constructedby combining phosphatidylethnolamine (PE) with one of a number of acidicamphiphiles. Externally triggered pH-sensitive liposomes combine anotherwise stable liposome with an external soluble component such as atitratable polymer or a titratable synthetic peptide which undergoes aconformational change upon acidification. To increase the efficacy ofintracellular delivery, liposomes can be made pH-sensitive and able tofuse with cellular membrane at decreased pH values (pH drop from 7.4 to6.5) or in the presence of polyethylene glycol. Some pH-sensitiveliposomes are composed of DOPE:Cholesterol hemisuccinate at molar ratios2:1.

The preferential targeting of a liposome composition of this inventionto the hair follicle can be optimized by the choice of phospholipids inthe liposome composition, and may depend additionally on the includedbeneficial compound. Optimization can be readily conducted by use of thein vitro histoculture assay method described herein by preparation andtesting of a panel of preselected liposome formulations according to thephospholipid parameters described herein.

Particularly preferred parameters for targeting beneficial compounds tohair follicles include the combined use of liposomes that have bothcationic lipids and are pH-sensitive.

Where the liposome composition will be lyophilized prior to storage forlater use, it is preferred to include a carbohydrate or carbohydrates ascryopreservative(s) to protect the integrity of liposomes in theresulting liposome composition during lyophilization and subsequentrehydration.

Cryopreservation relates to preserving the integrity of delicatesubstances when liquids containing them are frozen and dehydrated. Theuse of a carbohydrate as a cryopreservative of liposome integrity uponfreezing and subsequent lyophilization has been reported (Racker E.,Membrane Biol. 10:221-235, 1972; Sreter F. et al., Biochim. Biophys.Acta. 203:254-257, 1970; Crowe et al., Biochem. J. 242:1-10, 1987; Croweet al., Biochim. Biophys. Acta. 987:367-384, 1988.

Suitable carbohydrate cryopreservatives include trehalose, maltose,lactose, glucose and mannitol. According to a preferred aspect of thepresent invention, trehalose is included in aqueous buffer solution usedin the preparation of a liposome composition of the present invention(prior to lyophilization), preferably at a concentration in the range ofabout 50 mM to about 250 mM.

The phospholipids, which may be obtained from the manufacturer in anorganic solvent, are mixed together in the appropriate ratios to yieldthe specified composition. An antioxidant can also be added to reducealkyl chain peroxidation of the fatty acid portions of thephospholipids, and the organic solvent, if present, is removed byevaporation. One suitable antioxidant is butyrated hydroxy toluene.Preferably about 0.1% (by weight) of antioxidant is used.

The dried (evaporated) phospholipid mixture is then redissolved with anaqueous detergent solution. Suitable detergents include those which havea relatively high critical micelle concentration (CMC) (Womack et al.,Biochim. Biophys. Acta 733:210, 1983). Such detergents includedetergents having a CMC of greater than approximately 2 mM. Preferredare those detergents having a CMC of between approximately 2 to 25 mM.Such preferred detergents include 3-(3-cholamidopropyl)dimethylammonio!-1-propanesulfonate (CHAPS) andalkylglucopyranosides such as octyl beta-D-glucopyranoside, octylbeta-D-thioglucopyranoside and the like. Optionally, the detergentsolution may include other components. These components may includebuffer salts such as HEPES, Tris, phosphate, and the like; various othersalts such as NaCl, KCl, and the like; a carbohydrate cryopreservativesuch as trehalose, maltose, glucose, and the like; and glycine.

According to a preferred embodiment of the present invention, thedetergent solution comprises 20 mM Tris, pH 7.5, 150 mM NaCl, (TBS)containing 100 mM CHAPS, 150 mM trehalose and 0.8% glycine. According tothis preferred embodiment, the phospholipids are redissolved in thissolution to give a final concentration of about 20 mg/ml.

Purified proteins for use in a liposome, together with carrier protein,are combined with the redissolved phospholipids and the volume of theresulting mixture is adjusted with a buffer as described above,preferably containing cryopreservative (most preferably trehalose) andglycine but no detergent. Protein is admixed with carrier protein, suchas bovine gamma globulin, and sufficient buffer is added to adjust thefinal concentrations of active protein to 10 mg/ml, bovine gammaglobulin to 1 mg/ml, phospholipid to 4 mg/ml and detergent to 20 mM.Suitable buffers include TBS containing 150 mM trehalose and 0.8%glycine.

The resulting clear, colorless solution requires no vortexing orsonicating to ensure co-solubilization.

The detergent in the phospholipid admixture can be removed by a numberof methods resulting in a stable liposome composition having a proteinor nucleic acid associated with and inserted through the lipid bilayer.Suitable methods of removal of detergent include dialysis, tangentialflow diafiltration, cross flow hollow fiber filtration, treatment withhydrophobic chromatography resin, and simple dilution.

One preferred method of detergent removal from the phospholipidadmixture utilizes dialysis for at least 30 hours at room temperature indialysis membrane tubing against a buffer such as TBS containing 150 mMtrehalose, 0.8% glycine and 0.05% NaN₃ to remove the detergent. Anotherpreferred method of detergent removal utilizes resin treatment. Suitableresins include hydrophobic chromatographic resins such as AmberliteXAD-2 (Rohm and Haas Co. in Philadelphia, Pa.) or Bio-Beads SM-2 (BioRadin Richmond, Calif.). The resins may be used to remove the detergent,either by direct contact with the phospholipid solution admixture orseparated from it by a dialysis membrane. The rate of removal ofdetergent from the phospholipid admixture is proportional to the weightratio of the detergent in solution and the chromatographic resin beads.

The liposome solution resulting from the detergent removal step is thenmade to 5 mM CaCl₂. According to one preferred aspect, the liposomecomposition which contains the fully active compound is diluted to aconcentration of 50 mM Tris, pH 7.5, 75 mM trehalose, 0.8% glycine and10 to 15 mM CaCl₂ before use. Alternatively, the diluted reagent may belyophilized for long term preservation of its biological performancecharacteristics and then later reconstituted by suspension in waterbefore use.

Another preferred method of detergent removal avoids the use of eitherdialysis or resin treatment and yet provides for preparation of activereagent. According to this method, detergent solubilized phospholipidcompositions containing protein or nucleic acids are diluted into abuffer without detergent to produce mixed micelles containing thebeneficial compound which remain capable of being fully activated byCaCl₂. According to this aspect of the invention, phospholipids aredissolved to 20 mg/ml in a buffer containing detergent, preferably analkyl glucopyranoside. A suitable buffer-detergent solution comprises 20mM HEPES (pH 6) containing 50 mM octyl beta-D-thioglucopyranoside (OTG)and 150 mM NaCl. Carrier protein, active protein or nucleic acid, andCaCl₂ are then added and the mixture diluted further with buffer withoutdetergent, such as 20 mM HEPES (pH 6) containing 150 mM NaCl, to yieldfinal concentrations of active protein or nucleic acid at about 10mg/ml, carrier protein (bovine gamma globulin) at 1 mg/ml, CaCl₂ at 5mM, phospholipids at 4 mg/ml, and OTG at 10 mM. The reagent may belyophilized for storage as described above, or diluted as describedabove before use.

According to another aspect of the present invention, this reagent maybe prepared by following methods for the preparation of vesicles anddetergent-phospholipid mixed micelles from phospholipids by methodsbased on mechanical means, by removal of organic solvents, by detergentremoval, and by size transformation as has been described byLichtenberg, D. and Barenholz, Y., Methods of Biochemical Analysis, 33:337-462 (1988), and the disclosures of which are incorporated herein byreference.

Incorporation of a beneficial compound is conducted by incorporation ofthe compound in the liposome either during liposome formation, or afterformation by combining the liposome with the compound. Methods ofintroducing the compound into the liposome can vary, and are notintended to be limiting. Preferred methods are described in theExamples.

Where nucleic acid is entrapped into a phospholipid composition, a widevariety of ratios of nucleic acid to phospholipid may be utilized asdiscussed earlier. However, it is preferred to use about 100 micrograms(ug) of nucleic acid (in the form of double-stranded DNA such as plasmidDNA) with about 0.1 to 10.0 milligram (mg) phospholipid. Where cationicphospholipids are to be utilized in a phospholipid composition, it isparticularly preferred to use about 100 ug nucleic acid to from 0.2 to1.2 micromoles (umole) of phospholipid, particularly 100 ug nucleic acidto 0.8 umole.

Preferred liposome compositions of this invention comprise a liposomecontaining an effective amount of a beneficial compound of thisinvention. Preferred beneficial compounds depend upon the use of theliposome composition as described further herein, and can includemelanin, hair dyes, tyrosinase, nucleic acids, including sense andantisense nucleic acid molecules, hair color-restoring agents, hairgrowth-promoting agents, and agents which confer chemorestistance to thetargeted hair follicle.

3. Hair Follicle-Targeted Drug Therapy

Results of studies herein on liposome-mediated delivery show that thebeneficial macromolecular compound is concentrated at the hair folliclesand has been transported across the cell membrane and through thecytoplasm and in some cases to the nucleus. The liposome-incorporatedmaterial (beneficial compound) is preferentially delivered to the hairfollicle, because the levels of the beneficial compound in the adjacentskin tissue is substantially lower than in the hair follicles. Due tothe unusual selectivity of delivery to the hair follicle when using thedisclosed liposome formulations, and based on the degree of selectivitybased on the compound to be delivered and the liposome formulationutilized, this selectivity is referred herein to as "directed delivery","preferential delivery", "selective delivery" and in some cases as"exclusive delivery", depending upon the relative amount of materialdelivered to the hair follicle tissue as compared to the adjacent skintissue. In addition, the selectivity can be expressed in terms of theselectivity of pharmaceutical effect upon the hair follicle tissue ascompared to the adjacent skin tissue.

With the tissue sample treated with the macromolecular compound wherethe compound had not been incorporated in liposomes, very little reachesthe follicle cell or follicle cell nuclei. Thus, the liposome-basedsystem specifically, selectively, and efficiently targets the hairfollicles with compounds that otherwise do not concentrate at the hairfollicles.

In one embodiment, the invention describes methods for selective andbeneficial targeting of therapeutic compounds and compositions to thehair follicle of a mammal.

Based on the present disclosure, it is determined that compounds andcompositions, particularly polymers, dyes, proteins, nucleic acids andmacromolecules, are specifically delivered to hair follicle tissue, solong as the compounds or compositions are encapsulated in liposomes.

The invention contemplates the delivery of a wide variety of beneficialor otherwise therapeutic compounds to the hair follicle, with theselectivity of delivery to the hair follicle over adjacent skin tissuecells being of particular importance, and the primary result accordingto the present methods. Thus, the therapeutic compounds can be nucleicacids, hormones, proteins, small molecules, enzymes, steroids, vitaminsand other biochemical co-factors deemed to provide a therapeutic effectupon the hair follicle cell's growth, condition, color and the like.

Thus, a beneficial compound for use in the methods and compositions ofthis invention can be any of a variety of molecules including moleculesthat would not otherwise be able to reach hair follicles, such asmacromolecules and polymers that are too large to penetrate stratumcorneum or lipid barriers such as cell membranes, and lipophobicmolecules that are not able to penetrate lipid barriers due to theirchemical properties. Additional beneficial compounds for use in thepresent invention include lipophilic compounds that do exhibit acapacity to interact with and penetrate lipid barriers, but which canpenetrate other tissue barriers such as dermis where the compound canexhibit potentially undesirable effects upon cells external to the hairfollicle. Thus, a beneficial compound can be a macromolecule, a polymer,a lipophobic molecule, a lipophilic molecule having undesirable effectson cells external to the hair follicles, and the like compounds.

Particularly preferred are agents (beneficial compounds) which improvethe growth of the hair shaft, agents which stimulate the production ofhair coloring pigments in the hair follicle, agents which replacepigment in the follicle cell or hair shaft or which color (dye) the hairshaft (i.e., restore hair color), agents which stimulate hair growth,and agents which prevent hair loss (alopecia).

Agents useful for restoring or pigmenting hair color include melanin orhair dyes, which directly color hair as a pigment, the proteintyrosinase, which is an enzyme which catalyzes the production of melaninpigment precursors and thereby increases pigment production in hairfollicle cells, and nucleic acids which encode and express tyrosinaseand other proteins which stimulate hair growth or prevent hair loss. By"tyrosinase" is meant the protein tyrosinase or any derivatives,variants, analogs, or fragments thereof which are useful in the presentinvention. Such useful derivatives, variants, analogs, or fragments aredefined by their ability to catalyze the production of melanin pigmentprecursors. The term "fragment" includes any form of tyrosinase thatdoes not include the complete amino acid sequence of tyrosinase. Theterm "derivative" as used herein refers to a peptide or compoundproduced or modified from another peptide or compound of a similarstructure. This could be produced in one or more steps. The term"modified" or "modification" as used herein refers to a change in thecomposition or structure of the compound or molecule. However, theactivity of the derivative, modified compound, or molecule is retained,enhanced, or increased relative to the activity of the parent compoundor molecule. This would include the change of one amino acid in thesequence of the peptide or the introduction of one or more non-naturallyoccurring amino acids or other compounds. This includes a change in achemical body, a change in a hydrogen placement, or any type of chemicalvariation. In addition, "analog" as used herein refers to a compoundthat resembles another structure. An analog is not necessarily anisomer. The above are only examples and are not limiting.

As is well known, melanin is a polymer of tyrosine that occurs in avariety of forms and polymer lengths. Thus, the use of the term"melanin" is intended to mean melanin in any of its forms which can beutilized in the present invention. Also contemplated for use in thisinvention are derivatized melanin, extracted melanin, modified melanin,and the like variants of melanin which have the desirable property ofproviding hair pigment. Such variants include derivatives of melaninincluding compounds or polymers produced from tyrosine or modified fromanother compound, amino acid, or polymer of similar structure. The term"modified" or "modification" as used herein refers to a change in thecomposition or structure of the compound or molecule. However, theactivity of the derivative, modified compound, or molecule is retained,enhanced, or increased relative to the activity of the parent compoundor molecule. This includes a change in a chemical body, a change in ahydrogen placement, or any type of chemical variation.

Hair dyes are also extremely well known, and can take a wide variety offorms that need not limit the present invention. In particular, it isnoted that hair dyes are typically aromatic compounds which areincidentally mutagenic or otherwise exhibit undesirable effects ofvarious tissues of the body such as cells external to a hair shaft orhair follicle, such as in the dermis or in the circulation. Therefore itis to be emphasized that the present invention provides the advantage byvirtue of selective delivery to the hair follicle of reducing the extentof contact of administered hair dye with dermis and other tissuesexternal to the hair follicle.

Agents useful in conditions of hair loss (alopecia) are those whichstimulate hair growth, those which inhibit the hair loss, and thosewhich inhibit the conditions that cause hair loss, such aschemotherapeutic agents.

Hair growth stimulators are generally well known, and include minoxidil,substance-P, cyclosporin, cyclosporin A, finesteride, and the like knownhair growth stimulators.

Alopecia may be caused by numerous genetic and environmental factors,including but not limited to, stress, autoimmunity, and androgens.Agents useful in conditions of stress induced alopecia would be thosethat inhibit the delivery, to the cells in the hair follicle, of factorsthat cause the stress induced alopecia or those that protect thefollicle cells from such factors. Compounds that would be useful inpreventing hair loss caused by autoimmunity (alopecia areata) would bethose that inhibit the response of cells in the hair follicle toantibodies or other agents of the immune response (Sundberg et al., J.Invest. Dermatol. 104:32S-33S, 1995; Szafer et al., J. Invest. Dermatol.104:22S-24S, 1995). For example, such compounds would include, but arenot limited to, steroids which are known by those of ordinary skill inthe art to suppress the immune response. Another example of a compounduseful in the invention would be a compound that blocks the receptors oncells in the hair follicle which are targeted by the immune system inalopecia. Cell cycle inhibitors could also prevent alopecia caused bysuch agents or stress by keeping the hair follicle cells in the moreprotected G₁ -phase of the cell cycle.

An example of alopecia caused by androgens (androgenic alopecia) istypical male pattern baldness. The invention includes within its scopethe prevention of androgenic alopecia by delivery of compounds thatprevent androgenic alopecia to the hair follicle. Such compoundsinclude, but are not limited to, compounds that block the androgenreceptors on cells in the hair follicle such as steroids, steroidanalogs, androgen receptor antagonists, proteins, small molecules, ornucleic acid molecules.

In preferred embodiments, the delivered compounds are antisense nucleicacid molecules which hybridize to an androgen receptor gene, therebyinhibiting androgen receptor expression. In other preferred embodiments,androgenic alopecia is prevented by the selective delivery ofantiandrogens. By "antiandrogen" is meant a factor or molecule whichinterferes with the ability of an androgen, such as testosterone, toactivate an androgen receptor. Such antiandrogens include, but are notlimited to, 5-α-reductase inhibitors such as finesteride. 5-α-reductaseinhibitors interfere with the ability of testosterone to activateandrogen receptors by converting testosterone to dihydrotestosterone.The direct and selective delivery of antiandrogens and androgen receptorblockers to the hair follicle of the present invention advantageouslyavoids the side effects that may be caused by the presence of suchantiandrogens and androgen receptor blockers in the bloodstream and innon-hair follicle cells.

A preferred embodiment involves the prevention of hair loss (alopecia)during chemotherapy where a patient experiences chemotherapy-inducedhair loss due to the effect of the chemotherapeutic agent on the hairfollicle and surrounding tissue. Thus the invention contemplates the useof inhibitors of the deleterious effects of a chemotherapeutic agent. Byvirtue of the selective application of the inhibitor to the hairfollicle by the liposome-mediated delivery methods of the presentinvention, inhibition of a chemotherapeutic agent is localized to thehair follicle and therefore does not interfere with the intendedsystemic activity of the administered chemotherapeutic agent. In thisembodiment, a preferred inhibitor of chemotherapy-induced alopecia is agene product of the multiple drug resistance (MDR) gene, preferably thep-glycoprotein expressed by the human MDR-1 gene. Administration of anucleic acid comprising an expression vector capable of expressing humanp-glycoprotein via liposomes to the hair follicle provides intracellularhuman p-glycoprotein, and reduces the toxic effects of the chemotherapyupon the hair follicle, thereby reducing alopecia induced by thechemotherapy.

Another embodiment of the invention is an in vitro model of chemotherapyinduced alopecia. The in vitro model may be used to screen for compoundswhich may prevent chemotherapy induced alopecia. Such an in vitro systemmay also be used to screen for chemotherapeutics which do not causealopecia.

Thus, one aspect of the invention includes a method for inhibitingalopecia in a mammal comprising applying a therapeutically effectiveamount of a liposome composition to a skin area on a mammal having aplurality of hair follicles, said liposome composition comprising aliposome containing a therapeutically effective amount of a compoundcapable of inhibiting alopecia when delivered to said hair follicles andsaid liposome is capable of selectively delivering said compound to saidhair follicles.

In one aspect, the alopecia is androgenic. In another aspect, thealopecia is alopecia areata. In a preferred aspect, the compound is asteroid or a steroid analog.

In another aspect, the alopecia is induced by stress. Therapeuticcompounds useful for inhibiting stress-induced alopecia include, forexample, cell cycle inhibitors as described herein.

In one aspect, the alopecia-inhibiting compound delivered to the hairfollicles is a protein. In another aspect the alopecia-inhibitingcompound delivered to the hair follicles is a nucleic acid capable ofexpressing in cells of said hair follicles a protein that inhibitsalopecia.

Another embodiment contemplates the use of the human transformationgrowth factor-alpha (TGF-α) gene to reverse the "wavy" hair phenotype.See for example, Mann et al., Cell 73:249-261, 1993, and Luetteke etal., Cell 73:263-278 1993. Therefore the invention contemplates the useof a cDNA expression vector that expresses the TGF-α gene as abeneficial compound to reduce the incidence of wavy hair where thedeficiency of TGF-α gene is the cause of the wavy hair phenotype.

The invention additionally contemplates the administration of any genebeneficial to hair follicles. A gene is beneficial to hair follicleswhere it confers, upon selective delivery to the hair follicles by thepresent methods, a beneficial effect upon the hair follicle. Exemplarybeneficial genes include genes normally and preferentially expressed inhair follicle, and therefore important for normal gene function.Beneficial genes can be identified by any of a variety of molecularbiological methods. For example, a cDNA library of expressed genes canbe prepared from hair follicle tissue supporting healthy hair, and canbe enriched by subtractive hybridization against a cDNA library derivedfrom a non-hair-producing or vellus-hair-producing follicle tissue,thereby producing a library of cDNA molecules whose expression isspecific to hair follicles. Individual cDNA molecules from the hairspecific cDNA library can be further screened for therapeuticeffectiveness using the skin histoculture assay described herein.

Particularly preferred is a gene capable of stimulating hair growth,referred to as a hair growth stimulating gene. A hair growth stimulatinggene is any nucleic acid which stimulates hair growth uponadministration of the gene to hair follicles of skin according to thepresent liposome-mediated delivery methods. A hair growth stimulatinggene can be prepared from the hair specific cDNA library describedabove. The hair growth stimulating gene can be selected from the hairspecific cDNA library by a variety of methods. The gene can beidentified by subtractive hybridization using a cDNA library preparedfrom skin tissue which has vigorous hair shaft production against a cDNAlibrary prepared from skin tissue which is deficient in vigorous hairshaft production, such as patches of skin where hair is absent orthinning. Such areas of skin have hair follicles but the follicle cellsare experiencing changes in gene expression which affect the conditionof the hair, particularly the rate of hair shaft growth. The resultingcDNA library following subtractive hybridization against the hair growthdeficient cDNA library is further screened in the in vitro skinhistoculture assay for cDNA molecules capable of stimulating hair growthto identify hair growth stimulating genes. Methods for isolating cDNAlibraries and for conducting subtractive hybridization a well known inthe art, and are not to be considered as limiting to the presentinvention.

Agents that may be useful for regulation of hair growth, eitherdelivered directly or in the form of a nucleic acid which encodes anagent and is capable of expressing the agent in hair follicle cellsinclude, for example, parathyroid hormone-related protein and Bcl-2(Wysolmerski et al., Proc. Nat'l Acad. Sci. USA 91:1133-37, 1994; Holicket al., Proc. Nat'l Acad. Sci USA 91:8014-16, 1994; Hayman, et al., J.Pathol. 158:293-96, 1989; and Veis et al., Cell 75:229-40, 1993). Othersuch agents are known to those of ordinary skill in the art. Examplesincluded by way of example only are reviewed in Messenger, J.Investigative Dermatol. 101:4S-9S, 1993. An antisense nucleic acidmolecule against a gene that encodes a protein that inhibits hairgrowth, such as fibroblast growth factor-5 (FGF-5) is another example ofa potential selective liposome-mediated hair follicle therapy (Hebert etal., Cell 78:1017-25, 1994), hereby incorporated by reference herein.Thus, in an aspect of the present invention, a liposome compositioncomprising an antisense nucleic acid molecule against a hairgrowth-inhibiting factor is provided, as well as a method using such aliposome composition to regulate or stimulate hair growth. In preferredembodiments, the antisense nucleic acid molecule is directed againstfibroblast growth factor-5.

The therapeutic agent can be delivered to the hair follicle in the formof an active formulation, such as the pigmentation protein or enzymeitself, or can be provided through gene replacement therapy, where anucleic acid is introduced that expresses the protein to be delivered.In this mode, also referred to as gene therapy, a replacement therapyprotein is provided which exerts a beneficial effect. The protein isreferred to as a "replacement therapy" protein to connote that thetherapy administered is to reconstitute (replace) into the tissue aprotein-based function not previously present. It does not mean that agene or protein was first deliberately removed, and then replaced.

Again, some beneficial compounds may have the ability to exhibitundesirable effects on tissues or cells external to the hair follicle,such as the dermis or other tissues accessed by the general circulation.Therefore it is noted that the selectivity provided by the presentinvention provides the advantage of reducing toxicity or undesirableeffects of certain beneficial compounds. This is particularly importantfor cyclosporins useful as hair growth stimulators but which cansuppress the immune system is contacted with the circulation, and foragents which confer chemoresistance in tissues where it is undesirableto provide such resistance such as in the circulation.

A therapeutic amount of a therapeutic protein in a liposome compositionof this invention is an amount sufficient to produce the desired result,and can vary widely depending upon the disease condition and the potencyof the therapeutic compound.

Thus, in one embodiment, the invention contemplates a method fordirectly and selectively delivering a beneficial compound to the hairfollicles of a mammal comprising the steps of:

a) incorporating an effective amount of at least one selected beneficialcompound into a liposome; and

b) applying said liposomes to skin areas of the mammal having aplurality of hair follicles;

whereby said beneficial compound is preferentially transmitted to saidhair follicles and enters into said hair follicles.

In a related embodiment, the invention describes a method of directlyand selectively delivering a beneficial compound to hair follicles of amammal comprising the step of applying a liposome composition of thisinvention topically to skin areas of a mammal having a plurality of hairfollicles. The liposome composition comprises a liposome containing aneffective amount of at least one selected beneficial compound whereinthe liposome is capable of selectively delivering the beneficialcompound to the hair follicles and whereby the beneficial compound ispreferentially transmitted to the hair follicles and enters into thehair follicles.

As described, the beneficial compound can be a protein, a nucleic acidor other molecule having desirable properties upon delivery to the hairfollicle cell. Where the compound is a pigment such as melanin or hairdye, or is a protein, such as tyrosinase, the objective is to restorehair color in hair as demonstrated herein. Alternatively, the compoundcan be aromitase or cyclosporin where the objective is to stimulate hairgrowth. Alternatively, the compound can be a nucleic acid encodingtyrosinase, aromitase, p-glycoprotein, TGF-α, a hair growth stimulatinggene or other beneficial proteins, or can encode and express anantisense or ribozyme nucleic acid as discussed herein.

Thus, in a related embodiment, the invention contemplates a method forrestoring hair color to the hair of a mammal comprising applying atherapeutically effective amount of a liposome composition to a skinarea on the mammal populated with hair follicles. The liposomecomposition contains an effective amount of a beneficial compoundcapable of restoring hair color (a hair color-restoring agent). The haircolor-restoring agent can be any of a variety of hair dyes, the pigmentmelanin, the protein tyrosinase, or a nucleic acid capable of expressinga tyrosinase cDNA as described herein.

In a related embodiment, the invention contemplates a method forinhibiting chemotherapy-induced alopecia in a mammal undergoingchemotherapy comprising applying a therapeutically effective amount of aliposome composition to a skin area on the mammal populated with hairfollicles. The liposome composition contains an effective amount of abeneficial compound capable of inhibiting within the follicle cellenvironment the toxic effects of the chemotherapy, e.g., a protein thatconfers chemoresistance to hair follicle cells and hair follicles. Anycompound that inhibits chemotherapy toxicity is contemplated, althoughthe MDR-1 gene product (p-glycoprotein), or the MDR gene itself, isparticularly preferred.

In a related embodiment, a method is provided for inhibitingchemotherapy-induced alopecia in a mammal, comprising applying atherapeutically effective amount of a liposome composition to a skin areon the mammal having a plurality of hair follicles, wherein the liposomecomposition comprises a liposome containing a cell cycle inhibitor, andthe liposome is capable of selectively delivering the cell cycleinhibitor to the hair follicles. In a related aspect, the liposomecontains a nucleic acid capable of expressing in the cells of the hairfollicles a cell cycle inhibitor.

The introduction of chemotherapeutic agents has made a major impact onthe survival of patients with leukemia, lymphoma, and testicular cancer.Among the more common malignancies of adults, carcinoma of the breasthad been the most responsive to a wide variety of single agents andcombination programs (Harris et al., in Cancer: Principles & Practice ofOncology (Devita et al., Eds.) pp. 1119-1167, 1992).

Doxorubicin is one of the most commonly used drugs for breast cancertreatment and also the most alopeciagenic among the anticancer drugs(Hussein et al., Science, 249:1564-1566, 1990; Haynes et al., in CancerMedicine, (Holland, Ed.) pp. 2286-2290, 1993; Harris et al., in Cancer:Principles & Practice of Oncology (Devita et al., Eds.) pp. 1119-1167,1992; Weiss Semin. Oncol., 19:670-86, 1992). The mode of action for itsanti-cancer effect is still not certain. As a DNA intercalator, it hasbeen shown to induce topoisomerase II mediated DNA cleavages (Tewey etal., Science 226:466-468, 1984). Generation of free radicals has alsobeen proposed (Benchekroun et al., FEBS Ltrs. 326:302-305, 1993).

Cyclophosphamide (cytoxan), methotrexate (MTX), and 5-fluorouracil(5-FU) compose a combination (CMF) that is the most commonly used forbreast cancer. Cytoxan is an alkylating agent (Harris et al., in Cancer:Principles & Practice of Oncology (Devita et al., Eds.) pp. 1119-1167,1992). MTX is an antifolate; it is a competitive inhibitor ofdihydrofolate reductase (DHFR), the enzyme for maintenance ofintracellular reduced folate pools. Depletion of reduced folate poolsresults in cessation of DNA synthesis caused by lack of sufficient dTMPand purines (Fleming et al., Semin. Oncol. 19:707-719, 1992). 5-FU is anantimetabolite which blocks DNA precursor synthesis, and acts againstDNA and RNA (Cheson et al., Semin. Oncol. 19:695-706, 1992).

Alopecia is a common yet distressing side effect of many cancerchemotherapeutic agents and radiation therapy (Seipp, C A in Cancer:Principles & Practice of Oncology (DeVita et al., Eds.) pp. 2135-2136,1992; Cline, Cancer Nurs. 221-227, 1984; Merk et al., in Hair and HairDiseases (Orfanos et al., Eds) pp. 601-609, 1990). As patients embark onnew therapies, hair loss can induce a negative body image, alteredinterpersonal relationships, and often cause patients to rejectpotentially curative therapy (Hussein et al., Science, 249:1564-1566,1990; Cline, Cancer Nurs., 221-227, 1984).

The degree of hair loss is drug type dependent. Some agents are morecausative than others. Doxorubicin and cyclophosphamide, commonly usedfor breast cancer treatment are among the worst (Haynes et al., inCancer Medicine (Holland, Ed) pp. 2286-2290, 1993). Clinical trialsdocument a consistent 85-100% hair loss in patients receivingdoxorubicin (Blum et al., Ann. Intern. Med., 80:249-259, 1974; O'Bryanet al., Cancer, 1-8, 1973). Severe alopecia occurs in 75-90% of patientstreated with intravenous cyclophosphamide (Cline, Cancer Nurs. 221-227,1984). A large single dose of chemotherapeutic agent may cause hair lossimmediately or after 1 to 2 weeks. Smaller, multiple doses can causehair loss in 6-8 weeks. Radiation (>500 rad) also causes hair loss(Seipp, in Cancer: Principles & Practice of Oncology (DeVita et al.,Eds.) pp. 2135-2136, 1992; Haynes et al., in Cancer Medicine (Holland,Ed.) pp. 2286-2290, 1993). The severity of hair loss is dose dependent.Generally, the higher the dose, the better the therapeutic effect, andthe greater the alopecia. This process is usually reversible and usuallyrecovery is in one month after cessation of treatment (Seipp, in Cancer:Principles & Practice of Oncology (DeVita et al., Eds. pp. 2135-2136,1992; Haynes et al., in Cancer Medicine (Holland, Ed.) pp. 2286-2290,1993).

Several preventative methods have been proposed. Those include scalptourniquet, scalp hypothermia, or a combination of both, the rationaleof which is to reduce the blood circulation during chemotherapy (Cline,Cancer Nurs. 221-227, 19814; Dean et al., N. Engl. J. Med.301:1427-1429, 1979; O'Brien et al., N. Eng. J. Med. 283:1469, 1970).None of them have been shown to have a definite protective effect,although undesirable effects, such as headaches, may arise (Hussein,Science 249:1564-1566, 1990; Cline, Cancer Nurs. 221-227, 1984).

Hair follicles have a quite complex anatomical and differentiatingsystem. Matrix cells near the bottom of the follicle proliferate rapidlyand differentiate to become parts of the growing hair shaft. They areregenerated periodically from stem cells located in a bulge surroundingthe middle of the follicle. Also the system contains dermal papillacells at the base of the bulb and mesenchymal cells outside the folliclewhich provide necessary factors for hair growth. Additionally, inner andouter root sheath cells connect hair follicle to the skin, and sebaceouscells provide lubrication (Colasarelis et al., Cell 61:1329-1337, 1990;Messenger, J. Invest. Derm 101:2s, 1993; Lavker et al., J. Invest. Derm.101:16s., 1993).

Mammalian hair follicles undergo a life cycle. When they are producinghair they are in anagen (=growing phase of hair) and the matrix cellsproliferate rapidly. Then the follicle converts through catagen phase totelogen which is a resting state. After a month or so they arereactivated to start anagen again (Colasarelis et al., Cell61:1329-1337, 1990; Messenger J. Invest. Derm 101:2s, 1993; Lavker etal., J. Invest. Derm. 101:16s, 1993). It is well recognized that thehair follicle cycle is inherently programmed but can also be influencedby systemic factors that have yet to be identified (Lavker et al., J.Invest. Derm. 101:16s, 1993; Yuspa et al., J. Invest. Derm. 101: 27s,1993).

Living cells in hair matrix multiply more rapidly than those in anyother normal human tissue (Haynes, et al., in Cancer Medicine (Holland,Ed.) pp. 2286-2290, PA 1993). Dermal papilla cell lines have beenestablished (Lichti et al., J. Invest. Derm. 101:124s, 1993). Hairmatrix cells have been isolated and cultured in vitro (Reynolds et al.,J. Invest. Derm. 101:634-638, 1993). They are sensitive to cancerchemotherapeutic agents. Two mechanisms of alopecia caused by cancerchemotherapy have been recognized. The main one is that cytotoxic agentsinhibit a specific phase of the matrix cell to cause "anagen effluvium".Scalp hair is particularly sensitive because of rapid replication inanagen of about 85% of cells, especially those on the lower matrix. Areduction in matrix volume and mitotic rate in the hair bulb aftercyclophosphamide leads to a zone of diminished caliber in the hair,which then fractures easily. This process is dose dependent. At highdoses there is also telogen effluvium, a cessation of cell growth causedby damage to other follicle cells. Radiation, for example, causes both(Merk in Hair and Hair Diseases (C E. Orfanos et al., Eds.) pp. 601-609,1990; Tiemey et al., Br. J. Cancer 62:527-528, 1990).

Much less research effort has been spent on prevention of alopecia thanon protection from adverse effects on bone marrow and gastrointestinaltoxicity induced by antitumor agents (Hussein et al., Science249:1564-1566, 1990. Schuchter et al., Semin. Oncol. 19:742-751, 1992;Wood, N. Engl. J. Med., 312:1060, 1985). None of the current treatmentsfor drug induced alopecia based on reducing blood circulation in thescalp have been shown to give definite protective effects in breastcancer therapy besides having undesirable effects (Hussein et al.,Science 249:1564-1566, 1990; Seipp, in Cancer: Principles & Practice ofOncology (DeVita, et al., Eds.) pp. 2135-2136, 1992; Cline, Cancer Nurs.221-227, 1984; Merk, in Hair and Hair Diseases (Orfanos et al., Eds) pp.601-609, 1990; Jimenez, et al., Cancer Res. 2:5123-5125, 1992). Thisinvention represents a novel approach to this alopecia problem. Theexpected advantages of this approach are as follows: no interruption ofscalp blood circulation. The undesirable effects with the currenttreatment, such as headache (Cline, Cancer Nurs. 221-227, 1984) shouldbe eliminated and the current treatments will therefore be moreeffective. There is a limit to the modulation of blood supply to thescalp from the outside. And a complete cutoff of blood supply couldpotentially cause ischemia-reperfusion toxicity. In contrast, one canuse a potent antidote as desired and it can be kept on the scalp forseveral hours.

The present invention provides an in vitro and an in vivo model forchemotherapy-induced alopecia. An animal model for drug induced alopeciais indispensable to search for therapy. Experimental models in mice andmonkeys for hair disease are available (Malkinson et al., J. Invest.Derm. 101:135s-137s, 1993; Bazzano et al., J. Invest. Derm.101:138s-142s, 1993; Uno et al., J. Invest. Derm. 101:143s, 1993). YoungFischer or Sprague-Dawley rats develop alopecia, starting from the head,in around one to two weeks after treatment with ara-C, doxorubicin, orcytoxan (Hussein et al., Science 249:1564-1566, 1990; Jimenez et al.,FASEB 6:911-913, 1992; Jimenez et al., Cancer Res. 2:5123-5125, 1992).Alopecia induced by ara-C or doxorubicin was protected by systemicadministration of ImuVert, a biological response modifier and a membranevesicle-ribosome preparation derived from the bacterium Serratiamarcescens by a series of lytic and centrifugal steps (Hussein et al.,Science 249:1564-1566, 1990). Topical 1,25-dihydroxyvitamin D3 preventsalopecia induced by cytoxan or etoposide (Jimenez et al., Cancer Res.2:5123-5125, 1992).

The present invention provides a method of preventingchemotherapy-induced alopecia by delivering beneficial compounds, suchas cell cycle inhibitors, to the hair follicle. By temporarily stoppingthe growth of cells in the hair follicle causing the cells to convertfrom anagen to telogen, during the duration of chemotherapy, those ofordinary skill in the art will recognize that the cells in the hairfollicle will not be targeted by the chemotherapeutic, and hair losswill thus be prevented. Cell cycle inhibitors known to those of ordinaryskill in the art may be used to temporarily stop the growth of cells inthe hair follicle during the duration of chemotherapy. Cell cycleinhibitors known to those of ordinary skill in the art include, forexample, but are not limited to, p16, p15, p21, p27, and p28 (reviewedby Karp & Broder, Nature Medicine 1:309-320, 1995). At least one cellcycle inhibitor, p21, has been cloned and sequenced (Noda et al., Expt'lCell Res. 211, 1994). Other cell cycle inhibitors are described in, forexample, Paus et al., Brit J. Dermatol. 122:777-84, 1990; Chang et al.,Science 267:518-22, 1995; and Bertelsen et al., BIO/TECHNOLOGY13:127-131, 1995. Preferably, a nucleic acid molecule coding for a cellcycle inhibitor, in an expression vector capable of being expressed inhair follicle cells, is delivered to the hair follicle cells using themethods of the present invention.

Another aspect of the invention provides a method of screening forcompounds which increase or decrease chemotherapy induced alopecia,comprising the steps of explanting skin tissue into skin histoculture asdescribed herein, treating the skin histoculture with a liposomecomposition comprising the compound to be screened, having a controlskin histoculture, or treating the skin histoculture with a placebo,treating the skin histoculture with an alopecia-inducingchemotherapeutic drug, measuring alopecia in the skin histoculture byphysically detectable means, and comparing the skin histoculture treatedwith the compound to the skin histoculture treated with the placebo. Theplacebo may be, for example, either no treatment at all, a buffer usedfor the screened compound, or a compound known to not have any effect onchemotherapy-induced alopecia. The histoculture may be treated with thescreened compound either before or after treatment with thechemotherapeutic drug. A preferred method of screening in vitro is setout in the examples.

In another aspect of the invention is provided a method of screening forcompounds which increase or decrease chemotherapy induced alopecia invivo. This method comprises the steps of treating mammalian skin tissuewith a liposome composition comprising the compound to be screened orwith a placebo, treating the skin tissue with an alopecia-inducingchemotherapeutic drug, measuring alopecia in the skin tissue byphysically detectable means, and comparing the skin tissue treated withthe compound to the skin tissue treated with the placebo. A preferredmethod of screening in vivo is set out in the Examples.

The method can be practiced on a variety of mammals, includingagricultural stock such as cow, sheep, horse, goat, pig, and the like,pets such as cats, dogs or other domesticated mammals, and humans.Typically, the hair follicle is present in the skin of a mammal, and themethod is practiced in vivo on a living mammal for the purpose ofbenefitting the condition of the hair follicle or hair shaft of themammal.

In another embodiment, the compound delivered may be a chemotherapeuticdrug. Those of ordinary skill in the art are aware that certain types ofcancers, such as basal cell carcinomas, are caused by keratinocytes(Cotsarelis et al., Cell 61:1329-37, 1990). Thus, the present inventionprovides a means of directly and selectively delivering chemotherapeuticdrugs to treat the cancerous condition by delivering the drugs directlyto the keratinocytes in the hair follicle.

In another embodiment, the invention provides a method of directly andselectively delivering a compound comprising an agent that reduces skinwrinkles. Such agents may include, for example, collagen or elasticproteins. Such agents may be used to firm the skin and provide moresupport to the skin by strengthening the hair follicles.

In one embodiment, the selected beneficial compound is a protein whichaffects hair growth, alopecia, hair color or hair condition. Preferredare the proteins tyrosinase or aromitase, as well as nucleic acidscoding for hair modifying proteins. In a related embodiment, theselected beneficial compound is a pigment, such a melanin.

Melanin, hair dyes and tyrosinase are preferred for their role incoloring hair. Aromitase, minoxidil and cyclosporin-A are preferred fortheir role in stimulating hair growth. Other therapeutic compoundssuitable for use in stimulating hair growth in conditions of alopeciainclude cyclosporin analogs, substance P, estrogen analogs andanti-androgens. Therapeutic compounds suitable for use in preventinghair growth, such as facial or pubic hair, include alopecia inducers,catagen inducers, epidermal growth factor, and the like inhibitors ofhair growth.

In another embodiment, the selected beneficial compound is a nucleicacid capable of expressing a beneficial protein which affects hairgrowth, alopecia, hair color or hair condition as described earlier.Preferred are the nucleic acids that express the proteins tyrosinase,aromitase, or other hair-growth stimulators, the protein products of theMDR-1 gene (i.e., p-glycoprotein) to prevent chemotherapy-inducedalopecia, or enzymes which synthesize those proteins. Antisense nucleicacid molecules which target hair growth inhibitor genes such asfibroblast growth factor-5 may also be used (Hebert et al., Cell78:1017-25, 1994).

In one preferred embodiment, the invention contemplates a method forrestoring hair color in mammals, particularly man, in which the haircolor is greying for any of a variety of reasons, including age. Themethod comprises applying a therapeutically effective amount of aliposome composition of this invention to a skin area on the mammalhaving a plurality of hair follicles which exhibit fading or greyinghair color. The liposome composition preferably contains an effectiveamount of a hair color-restoring agent of this invention, such as a hairdye, melanin, tyrosinase or a nucleic acid capable of expressing humantyrosinase in the cells of the hair follicles.

In one embodiment, the application of the liposome composition can berepeated at defined intervals to provide prolonged effectiveness, suchas prolonged hair color-restoration or prolonged chemoresistancedepending on the treatment, as needed.

Insofar as a liposome composition of this invention is usedtherapeutically, the liposome composition is itself a therapeuticcomposition, and as such may also contain additional components.

Therapeutic compositions of the present invention contain aphysiologically tolerable carrier together with at least one species ofliposome composition of this invention as described herein, dispersedtherein as an active ingredient. In a preferred embodiment, thetherapeutic composition is not immunogenic when administered to a humanpatient for therapeutic purposes.

As used herein, the terms "pharmaceutically acceptable","physiologically tolerable" and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration upon a mammal or human without the production ofundesirable physiological effects such as nausea, dizziness, gastricupset and the like.

The preparation of a pharmacological composition that contains activeingredients dispersed therein is well understood in the art. Typicallysuch compositions are prepared as sterile compositions either as liquidsolutions or suspensions, aqueous or non-aqueous, however, suspensionsin liquid prior to use can also be prepared.

The active ingredient can be mixed with excipient which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. Suitable excipient are, for example, water, saline, dextrose,glycerol, ethanol or the like and combinations thereof. In addition, ifdesired, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like which enhance the effectiveness of the active ingredient.

The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein (e.g.,protein, nucleic acid or other compounds). Pharmaceutically acceptablesalts include the acid addition salts (formed with the free amino groupsof the polypeptide) that are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, tartaric, mandelic and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryof liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH value, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, propylene glycol,polyethylene glycol and other solutes.

Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, organic esters such asethyl oleate, and water-oil emulsions.

A therapeutic composition contains a liposome composition of the presentinvention, typically an amount of at least 0.1 weight percent ofliposome composition per weight of total therapeutic composition. Aweight percent is a ratio by weight of liposome composition to totalcomposition. Thus, for example, 0.1 weight percent is 0.1 grams ofliposome composition per 100 grams of total composition.

A therapeutically effective amount of a liposome composition, orbeneficial compound therein, is a predetermined amount calculated toachieve the desired effect, i.e., to effectively benefit the targetedhair follicle, depending upon the benefit to be conferred. Thus, aneffective amount can be measured by improvements in one or more symptomsassociated with the condition of the hair follicle or hair follicleshaft occurring in the patient.

Thus, the dosage ranges for the administration of the liposomecomposition of the invention are those large enough to produce thedesired effect in which the condition in the hair follicle to be treatedis ameliorated. The dosage should not be so large as to cause adverseside effects. Generally, the dosage will vary with the age, condition,sex and extent of the disease in the patient and can be determined byone of skill in the art.

The dosage can be adjusted by the individual physician in the event ofany complication.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's system to utilize the active ingredient, and degree oftherapeutic effect desired. Precise amounts of active ingredientrequired to be administered depend on the judgement of the practitionerand are peculiar to each individual. Typical dosage ranges are between50 μl to 200 μl/cm² skin surface area, preferably 100 μl/cm², ofliposome composition, prepared using a ratio of liposome:therapeuticcompound between 1:1 and 500:1, preferably about 4:1 to 10:1, morepreferably about 8:1 to 10:1. However, suitable dosage ranges forsystemic application are disclosed herein and depend on the conditionsof administration. Other regimes for administration are also variable,but may be typified by an initial administration followed by repeateddoses at one or more hour intervals by a subsequent administration.Regimes for administration of the liposome composition will depend onthe rate of hair growth and may vary according to the individualpatient. These periods may easily be adjusted by the patient'sphysician. Typical periods of administration range from once a day toonce every two weeks. Preferably, the liposome composition will beadministered from twice a week to once a week.

4. Nucleic Acid Expression Vectors for Gene Therapy

In a particularly preferred embodiment, the invention contemplates theuse of recombinant DNA molecules that can function as expression vectorsfor expressing a beneficial protein via a liposome-mediated targetingmethod of this invention.

"Recombinant DNA (rDNA) molecule" refers to a DNA molecule produced byoperatively linking two DNA segments. Thus, a recombinant DNA moleculeis a hybrid DNA molecule comprising at least two nucleotide sequencesnot normally found together in nature. rDNA's not having a commonbiological origin, i.e., evolutionarily different, are said to be"heterologous".

In living organisms, the amino acid residue sequence of a protein orpolypeptide is directly related via the genetic code to thedeoxyribonucleic acid (DNA) sequence of the structural gene that codesfor the protein. Thus, a structural gene or DNA segment can be definedin terms of the amino acid residue sequence, i.e., protein orpolypeptide, for which it codes.

An important and well known feature of the genetic code is itsredundancy. That is, for most of the amino acids used to make proteins,more than one coding nucleotide triplet (codon) can code for ordesignate a particular amino acid residue. Therefore, a number ofdifferent nucleotide sequences may code for a particular amino acidresidue sequence. Such nucleotide sequences are considered functionallyequivalent since they can result in the production of the same aminoacid residue sequence in all organisms. Occasionally, a methylatedvariant of a purine or pyrimidine may be incorporated into a givennucleotide sequence. However, such methylations do not affect the codingrelationship in any way.

The DNA segments for use in the present invention are characterized asincluding a DNA sequence that encodes a beneficial protein as describedherein. Particularly preferred segments encode tyrosinase, aromitase,other hair-growth stimulating proteins, melanin, p-glycoprotein, TGF-α,or enzymes that synthesize those proteins. That is, the DNA segments ofthe present invention are characterized by the presence of a structuralgene encoding one or more of the recited beneficial proteins. Preferablythe gene is present as an uninterrupted linear series of codons whereeach codon codes for an amino acid residue found in the beneficialprotein, i.e., a gene free of introns.

One preferred embodiment is a DNA segment that codes for an amino acidresidue sequence that defines a tyrosinase protein corresponding insequence to a wild type tyrosinase protein and the DNA segment iscapable of expressing tyrosinase. A preferred DNA segment codes for anamino acid residue sequence consisting essentially of the tyrosinaseencoding nucleic acid sequence. Human tyrosinase gene and its nucleotidesequence is well known including the cDNA sequence for expressing humantyrosinase, and has been described by Tamate et al., J. Exp. Zool.,250:304-311, (1980); Shibahara et al., J. Exp. Med., 156:403-414 (1989);Takeda et al., Biochem. Biophys. Res. Comm., 162:984-990 (1989);Bouchard et al., J. Exp. Med., 169:2029-2042 (1989); and Brichard, J.Exp. Med., 178:489-495 (1993).

Insofar as there is redundancy in the genetic code, it is understoodthat a variety of nucleotide sequences may be utilized to express aparticular amino acid residue sequence. Therefore, in one embodiment,the invention contemplates the use of a nucleotide sequence that encodesa human tyrosinase protein.

For expression of the human tyrosinase gene, any of a variety ofexpression vectors may be utilized so long as the vector is compatiblewith expression in mammalian cells, particularly human cells. Suitablevectors are well known. A preferred vector is the pRHOHT2 vectordescribed in the Examples, although other mammalian expression vectorsare suitable.

Another preferred embodiment is a DNA segment that codes an amino acidresidue sequence that defines a multiple drug resistance (MDR) geneproduct, preferably the MDR-1 gene product designated p-glycoprotein,corresponding in sequence to a wild type p-glycoprotein and the DNAsegment is capable of expressing p-glycoprotein. A preferred DNA segmentcodes for an amino acid residue sequence consisting essentially of thep-glycoprotein encoding nucleic acid sequence. Human p-glycoprotein, theMDR-1 gene and the MDR-1 nucleotide sequence are well known includingthe cDNA sequence for expressing human p-glycoprotein, and has beendescribed by Chen et al., Cell 47:381-389, 1986; Ueda et al., J. Biol.Chem. 262:505-508, 1987; and Kioka et al., Biochem. Biophys. Res. Comm.162:224-231, 1989.

Insofar as there is redundancy in the genetic code, it is understoodthat the invention contemplates the use of a nucleotide sequence thatencodes a human p-glycoprotein.

Another preferred embodiment is a DNA segment that codes an amino acidresidue sequence that defines a transforming growth factor-alpha (TGF-α)protein corresponding in sequence to a wild type TGF-α protein and theDNA segment is capable of expressing tyrosinase. A preferred DNA segmentcodes for an amino acid residue sequence consisting essentially of theTGF-α encoding nucleic acid sequence. Human TGF-α gene and itsnucleotide sequence is well known including the cDNA sequence forexpressing human TGF-α, and has been described by Jakowlew et al., Mol.Endocrinol. 2:1056-1063, 1988.

Insofar as there is redundancy in the genetic code, it is understoodthat the invention contemplates the use of a nucleotide sequence thatencodes a human TGF-α protein.

Homologous DNA and RNA sequences that encode the above beneficialproteins are also contemplated.

In another embodiment, the invention contemplates the delivery ofantisense or ribozyme nucleic acids to hair follicle cells for thepurpose of selectively inhibiting hair follicle gene expression, andcontrol aspects of hair follicle cell function.

Antisense nucleic acids are generally well known in the art and functionto hybridize with sense strands of messenger RNA (mRNA), therebyinterfering with the normal expression of the hybridized mRNA molecule.The sequence of the antisense nucleic acid depends, as is well known,upon the nucleotide sequence of the mRNA to be hybridized. See forexample, Stein et al., Science 261:1004-1012, 1993.

Ribozyme nucleic acids are also generally well known in the art assingle-stranded (ss) RNA molecules that are capable of selectivelycleaving ssRNA and ssDNA. The ribozyme is useful to selectively inhibitgene expression by cleavage of a target ssRNA or ssDNA molecule in ahair follicle cell.

Representative targets for antisense or ribozyme nucleic acids aredeleterious genes in hair follicle cells, such as the genes responsiblefor baldness, hair loss, loss of hair color, strength or condition, andthe like undesirable features of hair follicles and hair shafts. In apreferred embodiment, the invention contemplates liposome-mediateddelivery of an antisense or ribozyme nucleic acid capable of inhibitingexpression of the gene that produces androgen receptor, therebyinhibiting follicle cell production of the receptor, thereby reducinghair loss. Antisense nucleic acid molecules which target hair growthinhibitors such as fibroblast growth factor-5 may also be used (Hebertet al., Cell 78:1017-25, 1994).

The preparation and use of antisense or ribozyme nucleic acids is wellknown in the art, and the design of particular antisense or ribozymenucleic acids are not themselves considered to be part of the presentinvention. However, insofar as the invention contemplates methods forliposome-mediated delivery of antisense or ribozyme nucleic acids tohair follicles for the purpose of improving delivery and selectivity ofthe effect exerted by the delivered nucleic acid, the present inventionis not to be limited to any particular species thereof but ratherdescribes general methods of their delivery as a beneficial compound.

DNA segments (i.e., synthetic oligonucleotides) used to produce a largerDNA segment that encodes a beneficial protein can easily be synthesizedby chemical techniques, for example, the phosphotriester method ofMatteucci et al., (J. Am. Chem. Soc. 103:3185-3191, 1981, or usingautomated synthesis methods. In addition, larger DNA segments canreadily be prepared from smaller DNA segments by well known methods,such as synthesis of a group of oligonucleotides that define the DNAsegment, followed by hybridization and ligation of oligonucleotides tobuild the complete segment.

Furthermore, DNA segments consisting essentially of structural genesencoding a beneficial protein can be obtained from recombinant DNAmolecules containing a gene that defines the beneficial protein isolatedfrom natural sources. Exemplary natural sources are described in thereferences cited herein where the cDNA sequences are described.

In addition, the invention contemplates the use of a recombinant DNAmolecule (rDNA) containing a DNA segment of this invention. A rDNA canbe produced by operatively linking a vector to a DNA segment of thepresent invention.

As used herein, the term "vector" refers to a DNA molecule capable ofautonomous replication in a cell and to which another DNA segment can beoperatively linked so as to bring about replication of the attachedsegment. A vector capable of directing the expression of a gene thatencodes a beneficial protein is referred to herein as an "expressionvector". Thus, a recombinant DNA molecule is a hybrid DNA moleculecomprising at least two nucleotide sequences not normally found togetherin nature.

The choice of vector to which a DNA segment of the present invention isoperatively linked depends directly, as is well known in the art, on thefunctional properties desired, e.g., protein expression, and the hostcell to be transformed, these being limitations inherent in the art ofconstructing recombinant DNA molecules. However, a vector contemplatedby the present invention is at least capable of directing thereplication, and preferably also expression, of the beneficial proteinstructural gene included in DNA segments to which it is operativelylinked.

In preferred embodiments, a vector contemplated by the present inventionincludes a procaryotic replicon, i.e., a DNA sequence having the abilityto direct autonomous replication and maintenance of the recombinant DNAmolecule extrachromosomally in a procaryotic host cell, such as abacterial host cell, transformed therewith. Such replicons are wellknown in the art. In addition, those embodiments that include aprocaryotic replicon also include a gene whose expression confers drugresistance to a bacterial host transformed therewith. Typical bacterialdrug resistance genes are those that confer resistance to ampicillin ortetracycline.

Those vectors that include a procaryotic replicon can also include aprocaryotic promoter capable of directing the expression (transcriptionand translation) of the beneficial protein gene in a bacterial hostcell, such as E. coli, transformed therewith. A promoter is anexpression control element formed by a DNA sequence that permits bindingof RNA polymerase and transcription to occur. Promoter sequencescompatible with bacterial hosts are typically provided in plasmidvectors containing convenient restriction sites for insertion of a DNAsegment of the present invention. Typical of such vector plasmid arepUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories,(Richmond, Calif.) and pPL and pKK223 available from Pharmacia,Piscataway, N.J.

Expression vectors compatible with eucaryotic cells, preferably thosecompatible with mammalian cells, and particularly hair follicle cells,can also be used to form the recombinant DNA molecules for use in thepresent invention. Mammalian cell expression vectors are well known inthe art and are available from several commercial sources. Typically,such vectors are provided containing convenient restriction sites forinsertion of the desired DNA segment, and provide the signals requiredfor gene expression in a mammalian cell. Typical of such vectors are thepREP series vectors and pEBVhis available from Invitrogen (San Diego,Calif.), the vectors pTDT1 (ATCC #31255), pCP1 (ATCC #37351) and pJ4W(ATCC #37720) available from the American Type Culture Collection (ATCC)and the like mammalian expression vectors.

Particularly preferred are mammalian expression vectors which allow theexpression of the gene in a tissue-specific manner, in this case by theaction of a regulatory promotor that will limit gene expression to hairfollicle cells.

Successfully transformed hair follicle cells, i.e., follicle cells thatcontain a rDNA molecule of the present invention, can be identified bywell known techniques. For example, cells resulting from theintroduction of an rDNA of the present invention can be subjected toassays for detecting the presence of specific rDNA using a nucleic acidhybridization method such as that described by Southern, J. Mol. Biol.98:503, 1975, or Berent et al., Biotech. 3:208, 1985, or by in situhybridization techniques well known to those of ordinary skill in theart.

In addition to directly assaying for the presence of rDNA, successfultransformation can be confirmed by well known immunological methods forthe presence of expressed protein. For example, follicle cellssuccessfully transformed with an expression vector produce proteinsdisplaying beneficial protein, which then can be assayed directly byimmunological methods.

Alternatively, successful transformation of the target tissue can beconfirmed by evaluation of the target tissue for indicia of functionexerted by the administered beneficial compound. For example, where thecompound is a nucleic acid expressing tyrosinase, as described in theExamples, the exerted function of pigmentation, or the presence oftyrosinase activity or enzymatic conversion of L-dopa to product can bedetected directly in the target tissue.

B. Methods for Identifying Genes That Encode Proteins Beneficial to HairFollicles

In another embodiment, the invention provides a method for identifying agene that encodes a protein that can exhibit a beneficial effect upon ahair follicle. The method comprises the steps of (1) encapsulating anucleic acid molecule containing the gene of interest into a liposomecomposition of this invention, (2) contacting the nucleicacid-containing (encapsulated) liposome with a skin sample histocultureas described herein and having at least one hair follicle, therebydelivering the nucleic acid to the follicle, and (3) observing whetherthe delivered nucleic acid, upon expression of any protein encodedthereon, exhibits a beneficial effect on the hair follicle. The effectobserved can be changes in hair color, condition, growth rate,viability, condition of the associated hair follicle cell structures,and the like indicia of cellular response.

In one embodiment, the present method is well suited to screening genelibraries for the presence of a gene capable of expressing a proteinthat exhibits a beneficial effect on a hair follicle. Gene libraries canbe in the form of cDNA libraries or genomic DNA libraries as is wellknown. The beneficial effect to be induced depends on the screeningmethod to detect the effect, as described further herein.

The following Examples serve to illustrate particular embodiments of theinvention and are not limiting of the specification and claims in anyway. The examples detail the application and testing of liposome-basedtreatments hair follicles and hair growth problems. Parts andpercentages are by weight unless otherwise indicated.

EXAMPLES

1. Liposome-Mediated Delivery of Dye to Hair Follicles

Liposome-mediated delivery of beneficial agents to hair follicles isdemonstrated using a native-state skin sample histoculturing method inwhich hair follicle-containing skin samples are cultured allowing thegrowth of the hair follicle, and detailed observation of the hairfollicle cells during the treatment with therapeutic liposomes.

For the histoculture of skin, pieces of shaved outbredwhite-haired-mouse or nude-mouse skin, approximately 2×5×2 mm, wereharvested under a dissection microscope and then histocultured oncollagen-gel supported sponge as described by Li et al., Proc. Natl.Acad. Sci. USA 88:1908-1912, 1991. Histoculture was continued for about24 hours prior to contacting the skin histoculture with the liposomepreparation.

Liposomes were prepared by sonication of about 15 mg or 25 mg,preferably 25 mg, phosphatidylcholine (PC) emulsion in phosphatebuffered saline (PBS) containing about 20 mg/ml of the fluorescent dyecalcein. Liposomes were also prepared by entrappingNBD-phosphatidylcholine fluorescent dye using an emulsion with about 20mg/ml of the NBD formulation. Liposomes were separated from thenon-entrapped dye by gel-filtration on a Sepharose 4B column dilutedwith phosphate buffered saline. The amount of entrapped dye was measuredspectrofluorometrically. Two types of PC were used: egg PC (EPC) anddipalmitoyl PC (DPPC). Due to their phase transition temperatures,liposomes made of DPPC are in a gel phase at about 37° C. whileliposomes prepared from EPC are in a liquid-crystalline state.

Samples of the mouse skin histocultures were incubated for about 20minutes with each of the liposomes and with a solution of "free" calceindye at the same concentrations used in the liposome preparation. Afterthe tissue samples were thoroughly washed with culture medium free ofliposomes to remove excess liposome composition, the specimens wereanalyzed with a BioRad MRC 600 laser confocal microscope with a BHSfilter block, which excites the tissue at 488 nm and passes the lightemitted at 520 nm. These parameters are close to the excitation andemission maxima reported for calcein, Haugland, (Ed.) Molecular probes.Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes,Inc., Eugene, (1989-1991 and 1992-1993). There is no autofluorescence oftissue when these emission and excitation wavelengths are used. TheMRC-600 Confocal Imaging System (Bio-Rad, Richmond, Calif.) was mountedon a Nikon Optiphot equipped with a 10× PlanApo Objective.

FIG. 1A shows the skin histoculture incubated with calcein-entrapped EPCliposomes. Note the high efficiency of the delivery of the fluorescentdye preferentially into hair follicles. FIG. 1B shows the skinhistoculture incubated with free calcein solution. Note the relativelylow fluorescence with no preferential staining of any particular skinstructure. The image in FIG. 1B was made with the same parameters ofaperture and gain control as FIG. 1A.

To study the differences in liposome-mediated delivery depending uponthe type of liposome used, additional liposomes were prepared as aboveexcept using DPPC in the liposome. The results obtained using eithercalcein or NBD-phosphatidylethanolamine as the fluorescent label showedselective labelling of the hair follicle at the surface of the folliclerather than inside the follicles when EPC was used. Thus, differentliposome compositions allow even greater selectivity in delivery to apreselected region of the follicle.

The above results show a difference between substantially preferentialstaining of hair follicles obtained with dye entrained in DPPC or EPCliposomes compared to a lack of preferential staining of follicles overskin with free dye. Thus, liposome-entrapped dye, in contrast to freedye, becomes specifically associated with hair follicles, indicatingthat liposomes specifically target hair follicles. The results of thisexperiment have been confirmed in subsequent experiments.

FIG. 9 demonstrates another set of experiments of liposome delivery ofcalcein into the follicles in histocultured skin. FIG. 9A depicts mouseskin histocultures treated with calcein-entrapped PC liposomes for 48hours. Note the high efficiency of the delivery of the green fluorescentdye preferentially into the hair follicles and shafts. The sample hasbeen counterstained by propidium iodide (PI) to reveal the adjacentcells of the follicles. Note that no adjacent cells or epidermal cellswere stained with the green fluorescent dye. FIG. 9B demonstrates themouse skin histocultures treated with "free" calcein dye withoutliposomes which is at the same concentration as were entrapped into theliposomes in FIG. 9A for 48 hours. Note the very weak staining of a fewhair follicles.

FIG. 10 demonstrates liposome delivery of calcein into hair follicles inhistocultured skin by topical use. FIG. 10A shows the mouse skinhistocultures topically treated with calcein entrapped in eggphosphatidylcholine liposomes for 48 hours. Note that thecalcein-entrapped liposomes penetrated through the skin and deliveredcalcein dye into hair follicles. FIG. 10B is the control, topicallytreated with "free" calcein dye without liposomes which was at the sameconcentration as was entrapped into the liposomes in FIG. 10A for 48hours. Noted that the "free" calcein dye rarely penetrated through theskin to stain the hair follicles, whereas the liposomally-entrapped dyewas targeted very efficiently to the follicles.

2. Liposome-Mediated Delivery of Melanin to Hair Follicles

The targeted delivery of a beneficial compound to hair follicles wasdemonstrated using melanin as the model because melanin provides thebenefit of pigmentation.

To that end, liposomes were prepared by sonication. About 20 mg of eggphosphatidycholine was rotary evaporated with a vacuum drier from achloroform solution to form a thin film on the walls of a 5 mlround-bottomed flask for about 1 hour. The dried thin film phospholipidwas suspended in about 0.5 ml phosphate buffered saline (pH 7.4) on avortex mixer and then sonicated with a Branson probe-type sonicatorfitted with a microtip at power level 3 for about 8 minutes. Then 0.5 mlof a solution of melanin (10 mg/ml) was entrapped with the abovesuspension by sonication for about an additional 4 minutes. Liposomeswere separated from the non-entrapped melanin by gel-filtration on aSepharose 4B column equilibrated with phosphate buffered saline.

Pieces of outbred white-haired mouse skin derived from 1-2 weeks-oldanimals (about 2×5×2 mm each) were harvested under a dissectionmicroscope. The samples were then histocultured on collagen-gelsupported sponges as described in Example 1. Liposome interaction withthe skin was initiated after about 24 hours of histoculture. Mouse skinhistocultures were incubated for about 12 hours with liposomes. As acontrol, a solution of "free" melanin at the same concentration as wasused in the liposome preparation was also incubated for about 12 hourswith pieces of the histocultured skin.

The skin histocultures were counter-stained with the dye2',7'-bis(2-carboxyethyl)-5 which is activated to fluorescence bynonspecific esterases present only in living cells. After the tissueswere thoroughly washed, the specimens were analyzed with a Nikonfluorescence microscope equipped with a fluorescein cube.Microscopically the live tissues and cells fluoresced green such thatdark dense melanin deposits localized in the tissue can be clearlyidentified against the green background. All skin samples were thenfixed with formalin and processed through dehydration, paraffinization,paraffin-embedding and hematoxylin and eoxin (H&E) staining.

FIG. 2 shows the liposome-mediated targeted delivery of melanin to thehair follicles in the skin histocultures stained with H&E. In theparaffin section of white-haired mouse skin treated for 12 hours withthe melanin-entrapped liposomes, the majority of the melanin can be seento be localized around the hair follicles. The melanin can be seen atthe periphery of follicles and in the follicle cells themselves. FIG. 3shows a side view of a H&E-stained hair follicle, showing that theliposome-entrapped melanin was delivered into the hair shaft itself toform the band-like melanin-distribution pattern in theterminally-differentiated keratinocytes of the typical normal pigmentedhair shaft. Note that the liposome-delivered melanin seen in FIG. 3exhibits a natural pattern in the hair shaft mimicking a naturalmelanized hair shaft. In the control (not shown), in which the skinhistocultures had been incubated with the "free" melanin, no "free"melanin can be observed either in hair shafts or the hair follicularcells.

Thus, liposomes can specifically target an important, large, polymer tohair follicles and even enter into the hair shaft itself in a normalpattern.

In order to clearly demonstrate that the liposome preferentiallytargeted melanin to hair follicles, additional experiments wereperformed in which the samples were counterstained with propidium iodide(PI) to reveal the adjacent cells of follicles. As FIG. 11A demonstratesno adjacent cells were targeted by liposomes for melanin delivery. FIGS.11B & 11C demonstrate the histological sections of the same samples asFIG. 11A. Note that the melanin is only observed in the hair shafts andhair follicles. No epidermal and adjacent cells of the follicles containmelanin. Therefore we conclude that the liposomes are specificallytargeting melanin to the hair follicles.

Thus we have demonstrated that liposomes can specifically target animportant molecule, in this case melanin, to hair follicles inhistocultured mouse skin. The liposome-targeted melanin even enters thehair shaft itself in a normal pattern. Our results demonstrate the greatpotential for liposome-targeting of crucial substances to hair folliclesto modify the hair follicle and hair itself.

Mice were then treated with the melanin-containing liposome compositionsof the present invention in vivo, to produce gross coloring of thetreated mouse hair.

3-4 week old white-haired mice were depilated by a mixture of bees waxand rosin gum. Topical application of liposome-melanin as describedherein was started on day 6 after depilation. The liposome-melanincomposition was applied on the skin of mice once per day for 4 days. Theskin surface was then washed with an excess of water and wipe-dried witha gauze pad. The out-growing hair was micro-photographed. The sameconcentration of naked melanin solution as entrapped into liposomes wasused as the control.

FIG. 12A depicts the selective delivery of melanin to the hair folliclecells, the hair shaft itself is colored, while the surrounding skin isnot colored. No hair coloring is observed in the mouse treated withnon-liposome entrapped (naked) melanin, as depicted in FIG. 12B.

In another experiment, 3-4 week old white-haired mice were depilated bya mixture of bees wax and rosin gum. Topical application ofliposome-melanin composition was started on day 10 after depilation. Themelanin used in this experiment was from Biosource, SF, CA #MM-8A-1. Theliposome-melanin composition was applied on the skin of mice twice perday for 3 days. The treated skin surface was then washed with an excessof water and wipe-dried with a gauze pad. The mice were thenphotographed. The same concentration of naked melanin solution asentrapped into liposomes was used as the control.

FIG. 13B depicts the control mouse treated with naked melanin. Themouse's regrown hair remained white. In contrast, the regrown hair ofthe mouse depicted in FIG. 13A, that was treated with theliposome-melanin composition was colored, demonstrating selectivedelivery of melanin by the liposome-melanin compositions of the presentinvention.

3. Liposome-Mediated Delivery of Nucleic Acid to Hair Follicles

a. Delivery of Nucleic Acids to a Cultured Cell Line

The targeted delivery of nucleic acid to hair follicles was demonstratedusing mouse genomic DNA cleaved to about 1 kilobase (kb) lengths as themodel for nucleic acids capable of expressing protein due to the typicalsize of a DNA expression vector, and the size of a typical structuralgene.

About 1 kb DNA was isolated from a mouse genomic DNA library andpurified from low melting point agarose with the Magic DNA PurificationKit (Promega, Madison, Wis.). About 50 ng DNA was labeled with ³⁵ S!dATP(DuPont) with the Random Primer DNA Labeling Kit (BioRad, Richmond,Calif.). The specific activity of the labeled DNA with ³⁵ S-dATP was2.6×10¹⁰ cpm/μg.

Liposomes were prepared by freezing and thawing. About 20 mg of eggphosphatidylcholine (EPC) was rotary evaporated with a vacuum drier froma chloroform solution to form a thin film on the walls of a 5 mlround-bottomed flask for about 1 hour. The dried film phospholipid wassuspended in an about 0.5 ml phosphate buffered saline solution at a pHof about 7.4 in a vortex mixer and then sonicated with a Bransonprobe-type sonicator fitted with a microtip at power level 3 for about 8minutes. The 0.5 ml of ³⁵ S!dATP-labeled DNA solution was added to theabove suspension by extensive vortexing for about 1 minute and followedby freezing and thawing. Liposomes were separated from the non-entrapped³⁵ S!dATP by gel-filtration on a Sepharose 4B column equilibrated withPBS. About 50 μl calcein (about 10 mg/ml) was added into the solution inorder to mark the liposomes during the separation. The specific activityof the entrapped DNA labeled ³⁵ S!dATP was 2.5×10¹⁰ cpm/μl measured byliquid scintillation counting.

Pieces of outbred white-haired-mouse skin (about 1×5×2 mm) derived from1-5 week-old animals were harvested under a dissection microscope andthen histocultured on collagen-gel-supported sponges as described inExample 1. Liposome interaction with the skin was initiated after about24 hours of histoculture. Mouse skin histocultures were then incubatedfor about 44 hours with liposomes. As a control, a solution of naked- ₃₅S!DNA at the same concentration was used in the liposome preparation andwas also incubated with skin histocultures.

The skin histocultures were washed with phosphate-buffered saline, pH7.0, placed in histology capsules and fixed in 10% (v/v) formalin. Thefixed skin cultures were then dehydrated, embedded in paraffin,sectioned and placed on slides by standard methods well known to thoseof skill in the histology art. The slides were deparaffinized, coatedwith Kodak NTB-2 emulsion, exposed for 5 days and developed. See, e.g.Freeman et al., Proc. Soc. Natl. Acad. Sci. USA 83:2694-2698, 1986;Hoffman et al., Proc. Soc. Acad. Sci. USA 86:2013-2017, 1989; and Li etal., Proc. Soc. Acad. Sci. USA 88:1908-1912, 1991. The developed slideswere rinsed, stained with hematoxylin and eosin and examined using aNikon or Olympus photomicroscope fitted with epi-illuminationpolarization.

The histological autoradiogram of FIG. 4 shows ³⁵ S!DNA-labeled hair andfollicle cells in the histocultured skin after the skin was incubatedwith the DNA liposomes for about 44 hours. High radioactive labeling bythe ³ 5S!DNA in the cell membranes and cell cytoplasm as well as in thecell nucleus can be seen in FIG. 4, as pointed out by the arrows. Thisshows that the liposomes have delivered the DNA across the cell membraneand the DNA is transported through the cytoplasm to the nucleus.

When the histocultured skin was treated with naked- ³⁵ S!DNA there wereonly a few radioactive labelled cells. For further comparison, thepercent of labeled follicles per 20× field and percent of labeled cellsper follicle in the areas of maximum labeling can be calculated from theautoradiogram of FIG. 4. The percent of labeled follicles per 20× fieldis found to be about 7 times higher for the liposome carrying labeledDNA compared to the naked labeled DNA, and the percent of labeled cellsper follicle is at least 4 times higher for the liposome carried labeledDNA compared to the naked labeled DNA. For further comparison, we havecalculated from the autoradiograms the percent of labeled follicles per20× field and percent of labeled cells per follicle in the areas ofmaximum labeling. As can be seen in Table 1, both the percent of labeledfollicles per 20× microscope field and percent of labeled cells perfollicle in liposome- ³⁵ S!DNA-treated skin histocultures aresignificantly higher than in naked- ³⁵ S!DNA treated histocultures.

For further comparison, we have calculated from the autoradiograms thepercent of labeled follicles per 20× field and percent of labeled cellsper follicle in the areas of maximum labeling. As can be seen in Table1, both the percent of labeled follicles per 20× microscope field andpercent of labelled cells per follicle in liposome - ³⁵ S! DNA-treatedskin histocultures are significantly higher than in naked - ³⁵ S! DNAtreated histocultures.

                  TABLE 1                                                         ______________________________________                                        Liposome transfer of  .sup.35 S!DNA to hair follicles of                      histocultured skin.                                                                   Lipo- .sup.35 S!DNA                                                                          Naked- .sup.35 S!DNA                                   ______________________________________                                        Percent of                                                                              37.50 (6/16)                                                                             5.41 (2/37)                                                                             p < 0.05                                       labeled follicles                                                             per 20X field                                                                 Percent of                                                                              51.06 (24/47)                                                                            9.30 (4/43)                                                                             p < 0.005                                      labeled cells                                                                 per follicle                                                                  ______________________________________                                          .sup.35 S!DNA was entrapped in PC liposomes as described above. The          liposomes were incubated with the skin histocultures described above.         Naked  .sup.35 S!DNA was used as a control. Follicular  .sup.35 S!DNA was     analyzed by histological autoradiography in the areas of maximum labeling                                                                              

This Example demonstrates that liposomes can specifically andefficiently target DNA into the hair follicles, and therefore establishthat liposome encapsulated nucleic acids are useful reagents fortargeting gene therapy to hair growth processes.

b. Delivery of Liposome-Entrapped Nucleic Acid Expressing the HumanTyrosinase Gene to a Cultured Cell Line

Cloned human tyrosinase gene was transferred to tissue cultured celllines using liposomes to demonstrate the efficiency of liposome-mediateddelivery and expression of a tyrosinase gene.

To that end, liposomes were prepared by well known freezing and thawingmethods. About 20 mg of phospholipid in a ratio of 5:3:2 ofphosphatidylcholine (PC):cholesterol (Chol):phosphatidylethanolamine(PE) was rotary evaporated with a vacuum drier from a chloroformsolution for 1 hour to form a thin film on the walls of a 5 mlround-bottomed flask for about 1 hour. The dried film phospholipid wassuspended in 2 ml phosphate buffered saline solution at a pH of about7.4 (PBS) in a vortex mixer and then sonicated with a Branson probe-typesonicator fitted with a microtip at power level 3 for about 8 minutes.Then 200 ug of the plasmid pRHOHT2 was entrapped in a liposome byaddition of the plasmid to the sonicated suspension, sonication of theadmixture in a water bath for 2 minutes, followed by freezing andthawing three times to form nucleic acid-containing liposomecomposition.

Plasmid pRHOHT2 was obtained from Dr. S. Shibahara and is described byShibahara et al., J. Biol. Chem. 262:12889-12892, 1987, and Takeda etal., Biochem. Biophys. Res. Comm. 162:984-990, 1989, and contains a fulllength human tyrosinase cDNA, including promoters for expression oftyrosinase in mammalian cells.

Human fibroblast FS-3 and mouse amelanotic K1735 cell lines were eachpre-cultured in 60 mm culture dishes with Eagle's MEM medium containing10% fetal bovine serum (FBS) and Dulbecco's Modified Eagle's mediumcontaining 10 FBS, respectively, for 24 hours. Thereafter, the culturedcells were contacted with 0.5 ml of the tyrosinase gene-entrappedliposome composition in 1.5 ml of the respective culture medium perculture dish, and the contacted cells were maintained for 48 hours underculturing conditions. Thereafter, the cells were further cultured for 7days (FS-3) or 3 days (K1735) with normal culture medium afteraspiration of the liposome-containing medium. The cells were thenharvested by trypsin digestion and centrifuged at 800×g for 5 minutes toattach the cells to cytospin slides. As a control, 50 micrograms (ug) ofnaked plasmid in 0.5 ml medium was added to the two cell types in placeof the 0.5 ml liposome preparation.

The expression of tyrosinase was evaluated by measuring dopa-oxidasereactions and immunohistochemical staining for tyrosinase in the treatedcells.

To detect dopa oxidase activity, the cytospin slides were incubated with1 mg/ml of L-dopa in PBS for 12 hours at 37 degrees C. as described byKugelman et al., J. Invest. Dermatol. 37:73-76, 1961. Thereafter, thecytospin slides were counterstained with hematoxylin and eosin byestablished procedures, and the dopa oxidase-positive cells wereidentified and counted using a microscope.

To detect tyrosinase immunohistochemically, a Dako LSAB (labeledstreptavidin-biotin) kit was used to stain tyrosinase-containing cells.The cytospin slides were fixed in acetone for 10 minutes, and thenair-dried. Thereafter, serial incubations were then performed for 10 mineach sequentially in hydrogen peroxide, blocking serum, a dilution(1:400) of primary antibody (anti-tyrosinase), linking antibody,peroxidase-conjugated streptavidin, and 3-amino-9-ehtylcarbazolsubstrate solution as described by the manufacturer of the kit (Dako,Carpinteria, Calif.) . The primary antibody was rat anti-humantyrosinase monoclonal antibody TMH1 described by Tomita et al., J.Invest. Dermatol. 85:426-430, 1985, and Jimenez et al., Proc. Natl.Acad. Sci. USA 85:3830-3834, 1988. The linking antibody was a mixture ofanti mouse and anti rat IgG conjugated to biotin, provided by themanufacturer (Dako). The treated cytospin slides were then lightlycounterstained with Mayer's hematoxylin and mounted with liquid glycerolgelatin (Dako). A positive control was similarly prepared using a frozensection of human melanoma tissue. A negative control was prepared byreplacing the primary antibody with PBS.

The results show tyrosinase expression in both FS-3 and K1735liposome-treated cells, when detected by either dopa oxidase reaction orby immunohistochemical staining. The percent of cells expressingtyrosinase was approximately 52% of the total cells, by either assaymethod. The negative control cells were negative for both the oxidaseassay and the immunohistochemical staining assay.

When compared to the calcium phosphate method for transfection ofnucleic acid into cultured cells, it was observed that efficiency oftransfer of a tyrosinase-gene expression plasmid into cells was about 50times greater when liposomes were used in comparison to calciumphosphate.

These results demonstrate that liposomes are effective and efficient atdelivering nucleic acid expression vectors into cells, and further thatthe liposomes can deliver expression vector plasmid which aresubsequently able to express the encoded gene. Finally, the resultsdemonstrate that the tyrosinase gene can be effectively introduced andexpressed in mammalian cells.

c. Delivery and Expression of Beta-Galactosidase Gene in Hair Folliclesof Histocultured Skin

The bacterial gene Lac-Z encoding beta-galactosidase was delivered tohistocultured skin samples in a liposome preparation to demonstrateselective delivery and expression in hair follicles. PlasmidpM-MuLV-SV-Lac-Z contains a mammalian promoter derived from the Moloneymurine leukemia virus (M-MuLV) and the SV40 Virus (SV) which controlsthe expression of the beta-galactosidase gene (Lac-Z) capable ofexpression of beta-galactosidase in mammalian cells.

Liposomes were prepared as described in Example 3b, except that thephospholipids comprised PC, PE and cholesterol in a ratio of 5:2:3, andthe ratio of plasmid DNA to phospholipid was 200 ug DNA per 20 mg totalphospholipid.

White-haired mouse skin was histocultured as described in Example 1,except that the liposome composition was maintained in the culturemedium for four days. Thereafter, the skin histoculture medium waschanged to the same medium lacking liposomes and including the Lac-Zsubstrate X-gal, and the X-gal-containing medium was maintained underhistoculturing conditions for 18 hours to allow any beta-galactosidasepresent in the histocultured skin sample to convert the X-gal to thetypical visible blue dye. Control liposome delivery was conducted withnaked plasmid DNA (pM-MuLV-SV-Lac-Z) using the same amount of DNA aswith the liposome-entrapped plasmid composition.

Histocultured skin samples were then sectioned for histochemistry andevaluated using light microscopy at 125× and 250× magnification. Theresults are shown in FIGS. 5A-5D. The presence of expressed Lac-Z geneindicated by dark blue spots is only seen in FIGS. 5A and 5B whichreceived liposome-entrapped plasmid; no dark spots are observed in FIGS.5C and 5D. Furthermore, the dark spots are observed in the hairfollicles and not significantly observable in the tissues adjacent tothe hair follicles.

The results show that the Lac-Z gene was expressed in hair follicles andwas not detectable in the other portions of the histocultured skinsample, indicating the selectivity of the liposome delivery method.

4. In Vivo Liposome-Mediated Delivery of Beneficial Compounds to HairFollicles in Mice

The present methods were used to deliver beneficial compounds to hairfollicles in vivo by administration of a liposome composition of thepresent invention containing either melanin or calcein, or nucleic acidto mice.

a. Delivery of Melanin or Calcein to Hair Follicles in Mice

Liposomes were prepared essentially as described in Example 1. Twentymilligram (mg) of PC were rotary evaporated as described, andresuspended by sonication in 0.5 ml of PBS. Thereafter, 0.5 ml of eithercalcein (10 mg/ml) or melanin (10 mg/ml) solution, respectively, wereadded to sonicated PC liposome composition, and further sonicated for 6minutes, followed by freeze-thawing three times. The resulting liposomecomposition was extruded through a 0.6-1.0 uM filter and separated fromthe non-entrapped calcein or melanin by gel filtration on a Sepharose 4Bcolumn eluted with PBS to form liposome-entrapped beneficial compoundcomposition (calcein or melanin).

Two to 4 week-old pre-shaved outbred white-haired mice were used for invivo topical liposome delivery of entrapped beneficial compound to hairfollicles. A sample of about 250 microliters of the liposome compositionentrapping calcein or melanin was applied directly to the dorsal skin onthe mouse in an area of approximately 1.5 cm² using a sutured band-aidpatch to immobilize the liposome composition onto the skin and toprevent evaporation. The liposome composition was re-applied every 1hour for 6 hours, with the last application remaining for a total of 24hours at which time the skin samples were taken by punch biopsy foranalysis. For time course experiments, one mouse was used for each timeperiod, with 6 punch biopsies taken from each mouse, at 0.5, 1, 2, 4, 6,16 and 24 hours. Prior to punch biopsy, the skin was cleaned with analcohol swab to eliminate any material remaining on the surface of themouse's skin. For controls, the same amount of calcein or melanin wasapplied without liposome as with samples containing liposome-entrappedbeneficial compound.

After liposome treatment, the skin samples were harvested and cut tovery thin (5 mm) pieces of tissue sectioned along the vertical directionof the hair follicles, and subsequently observed by either light orfluorescent microscopy and photographed. For melanin-treated samples,the tissue sample was first counter-stained with BCECF-AM and propidiumiodide (PI) for fluorescent microscopy, or prepared for histology andstained in paraffin sections using hematoxylin and eosin for lightmicroscopy. For calcein-treated samples, the tissue sample was firstcounter-stained with propidium iodide (PI) for fluorescent microscopy.

Skin samples containing calcein were also analyzed by spectrofluorimetryto determine the effective concentration of a delivered beneficialcompound into a selected tissue. To that end, three samples eachcontaining two pieces of a 2-mm punch biopsy of skin for each time pointwere put into 2 ml of PBS and sonicated in the water bath sonicator for2 min. The sonicated sample was then centrifuged for 10 minutes in amicrocentrifuge at 14,000×g, and the resulting supernatant was measuredby spectrofluorimetry at an excitation wavelength of 496 nm and anemission wavelength of 517 nm for detecting calcein. The concentrationof calcein delivered into the skin tissue was determined from thespectrofluorometric readings by comparison to a standard curve.

FIGS. 6A-6C show the results of delivery of calcein usingliposome-entrapped calcein (FIGS. 6A and 6B) and naked calcein (FIG. 6C)after 20 hours. Notice that the liposome-mediated delivery has allowedthe calcein to penetrate deep into the hair follicles and shafts,whereas control calcein was trapped in the stratum corneum and did notenter the hair shafts or follicles.

Time course analysis of effectiveness of liposome-entrappedcalcein-mediated delivery showed that by 24 hours 22.15 nanograms (ng)per mm² of calcein was observed delivered in the hair follicles, whereasonly about 1.4 ng/mm² of naked calcein was observed delivered after 24hours, and this amount did not increase with time.

FIGS. 7A-7C illustrate the results in which melanin has been deliveredusing liposome-entrapped compound after 24 hours of treatment. FIGS. 7Aand 7B show the melanin delivered into the hair shafts in a pattern thatillustrates that the delivered melanin forms the exact pattern ofnaturally melanized hair shafts. FIG. 7C shows the melanin deliveredinto the hair follicle cells. Therefore, these results demonstrate thattopical in vivo administration of liposome resulted in delivery ofmelanin to both the hair follicle and hair shaft.

Skin samples containing melanin were also analyzed by spectrophotometryto determine the effective concentration of a delivered beneficialcompound into a selected tissue. To that end, three samples eachcontaining two pieces of a 2-mm punch biopsy of skin for each time pointwere put into 2 ml of PBS and sonicated in the water bath sonicator for2 min. The sonicated sample was then centrifuged for 10 minutes in amicrocentrifuge at 14,000×g, and the resulting supernatant was measuredby spectrophotometry at an absorption wavelength of 300 nm for detectingabsorption by melanin. The concentration of melanin delivered into theskin tissue was determined from the spectrophotometric readings bycomparison to a standard curve.

Readings from the skin samples in the time course study showed thatliposome-entrapped melanin was delivered specifically to the hairfollicles after 16 hours to a level of about 19.0 ug/mm² whereas lessthan 2.0 ug/mm² of melanin was delivered in the same time period usingnon-entrapped melanin.

The above results indicate that the liposome-targeted melanin or calceinwere selectively delivered to the hair follicle and hair shafts withinthe follicle, but non-entrapped compound was not delivered to the hairfollicle, and instead was restricted to the skin surface, particularlythe stratum corneum. Therefore, the liposome-mediated delivery ofbeneficial compounds is seen to be effective at specific delivery tohair follicles and hair shafts in a living animal, demonstrating in vivoefficacy of the liposome-mediated delivery methods described herein.

As a further control plasma calcein concentrations were also measured bytaking blood samples from the lateral tail vein of mice during thecourse of administration of liposome-entrapped calcein at 0.5, 1, 2, 4,6, and 24 hours after topical administration. The harvested blood wastransferred to a serum separator tube (Vacutainer, Becton Dickinson),and spun at 2000×g for 10 min to isolate plasma. Thereafter, calcein wasmeasured by spectrofluorimetry at an excitation wavelength of 496 nm andan emission wavelength of 517 nm for detecting calcein. Theconcentration of calcein in the plasma was determined from thespectrofluorometric readings by comparison to a standard curve. Over a24 hour time period, no detectable calcein entered the bloodcirculation. This is an important observation as it indicates that abeneficial compound, when administered by the present liposome-mediatedmethods, can be selectively targeted to the hair follicle and hair shaftwithout entry into the systemic circulation where it may exertundesirable side effects, and that safe follicle/shaft delivery ispossible.

b. Delivery and Expression of Beta-Galactosidase Gene in Hair Folliclesof Mice

1. Purification of Lac-Z DNA

A recombinant retrovirus containing the Lac-Z gene (pM-MuLV-SV-LacZ) wasobtained from Dr. Joshua R. Sanes (Washington University, St. Louis)(Sanes et al., EMBO J. 5:3133-42, 1986). Other expression vectorscomprising the Lac-Z gene may also be employed, such as pCH110, whichcontains the SV40 early promoter (Pharmacia Biotech). The plasmid Lac-Zwas transformed to HB101 E. coli competent cells (Promega) by standardmethods. The purification of plasmid Lac-Z DNA was obtained by using thePromega Wizard™ Megaprep DNA purification system.

2. Preparation of Lac-Z-liposomes

A total of 20 mg of lipid in a ratio of 5:3:2 of phosphatidylcholine(PC):cholesterol (Chol):phosphatidylethanolamine (PE) were rotaryevaporated for 1 hour with a vacuum drier from a chloroform solution toform a thin film on the wall of a 5 ml round-bottomed flask. The driedthin film lipid was suspended in 0.6 ml Tris-EDTA (TE) buffer containingapproximately 1 mg of Lac-Z DNA on a vortex mixer. The Lac-Z DNA wasentrapped by sonication in a compact water bath sonicator for 20minutes, followed by freezing (at -70° C.) and thawing (at roomtemperature) three times.

3. Topical application of Lac-Z-liposomes

Pre-shaved 5-6 week old Balb-c mice were used for the hair follicle genetherapy experiment. The skin area for application of liposome-Lac-Z waspre-hydrated with phosphate-buffered saline (PBS) for 10-30 minutes. 50μl of liposome-Lac-Z formulation were directly put on the skin area withreapplication after one hour. Untreated mice and mice treated with nakedLac-Z DNA mice were used as controls. The skin was carefully cleaned by70% isopropyl alcohol before harvest for X-gal staining 3 days afterapplication of the liposome-Lac-Z formulation.

4. Detection of Lac-Z DNA Expression by X-gal Staining.

The harvested skin samples were immediately put into a MEM wash mediumcontaining a combination of antibiotics at 4° C. for 1 hour and thenfixed in 2% (v/v) formaldehyde-0.2% (v/v) glutaraldehyde in PBS for 30minutes at 4° C. The tissues were then rinsed with PBS three times andincubated in the X-gal staining solution containing 1 mg/ml X-gal, 5 mMpotassium ferricyanide, 5 mM potassium ferrocyanide, and 2 mM MgCl₂ inPBS, at 37° C. for 18 hours. Skin tissues were processed for paraffinsectioning by standard histological procedures and photographed underlight microscopy after counter-staining with 0.1% nuclear fast red.

FIG. 14 illustrates the results of topical application of liposome Lac-Zto mice. Blue stain indicates gene activity in the hair follicles afterstaining with the X-gal substrate. Note the selective delivery of theactive gene to the hair matrix cells in the follicle bulbs (a-c) andalso to what may be follicle stem cells in the bulge area (d & e). Notethe high frequency of transfection of Lac-Z gene in the hair follicles(a) and also total lack of gene activity outside the hair follicle (a &b). Nuclear fast red counter-staining. Light Microscopy. Magnification200× (a), 400× (b), 1000× (c,d,e).

FIG. 14 shows that after topical application of liposome-Lac-Z, theexpression of the Lac-Z gene, indicated by blue staining of the X-galsubstrate, was in the hair-forming hair matrix cells in the hairfollicle bulbs (FIG. 14 a-c) and in the bulge area (FIGS. 14d & e) belowthe opening of the sebaceous gland, which is thought to contain thefollicle stem cells (Cotsralelis et al., Cell 61:1329-1337, 1990). Thetransfection frequency was high since many follicles are stained byX-gal (FIG. 14a). No other cells were transfected with Lac-Z outside thefollicle in the dermis or epidermis (FIG. 14a). FIG. 14b demonstratesthe expression of the Lac-Z gene in the hair matrix cells. The extensiveLac-Z expression in the hair matrix cells can be seen very clearly athigh magnification (FIG. 14c). The transfection of what may be folliclestem cells can be seen in FIGS. 14 d & e. The introduction of activegenes in stem cells is important for long-term modification of the hairfollicle. Topical application of the naked Lac-Z gene did not result ingene transfer and no Lac-Z staining can be seen in follicles in animalsnot treated with liposome-Lac-Z (data not shown). These resultsdemonstrate that genes can be selectively targeted to the most importantcells of the hair follicle by liposomes. The targeting of the reportergene to the hair follicle cells is the most selective targeting of agene observed thus far in vivo (Caplen et al., Nature Med. 1:39-46,1995). The high hair-follicle selectivity of gene targeting by topicalliposome application suggests the feasibility of gene targeting of hairmatrix cells and possibly follicle stem cells to restore hair color suchas with the tyrosinase gene (Shibahara et al., J. Exp. Med. 156:403-405,1988; Tanaka et al., Development 108:223-227, 1990) and with genes torestore hair growth. The high selectivity of topical liposome genetargeting is also important for safety and cosmetic reasons.

5. Screening for Compounds which Increase or DecreaseChemotherapy-Induced Alopecia

a. In Vitro Model of chemotherapy-Induced Alopecia

5-day old Sprague-Dawley rat skin tissues (4 mm size punch) areexplanted on the collagen-containing gels and histocultured for 3 days.The skin histocultures are then treated with melphalan (40ug/ml)+doxorubicin (1.16 ug/ml) for 4 days. To screen for compounds thatprevent alopecia in vitro, the skin histocultures are pretreated withliposomes containing the compound as described in Example 1, (100 μg/ml,1 mM β-ME) for 2 days after an initial 24 hours histoculture.Liposome-compound treatment is maintained during chemotherapy for 4 daysof histoculture. Four pieces of skin tissue are used in each group.Control is skin histoculture without pre-treatment and chemotherapy. Theeffect of chemotherapy on hair loss and prevention by the compound inhistocultured skin was determined by dissection microscopy.

Cell cycle inhibitors are an example of compounds that may be used toprevent chemotherapy-induced alopecia. An example of a cell cycleinhibitor, p21, has been tested by the present inventors and has beenfound to prevent chemotherapy-induced alopecia. An expression vectorcomprising a nucleic acid molecule coding for p21 (Noda et al., Expt'lCell Res. 211, 1994) was entrapped in a liposome composition accordingto the methods of the present invention. The liposome formulationcontained a ratio ofphosphatidylcholine:cholesterol:phosphatidylethanolamine of 5:3:2,however, any of the liposome compositions of the present invention, maybe used. The ratio of lipid:p21-expression vector was approximately10:1, this ratio may vary, as described herein.

The p21/liposome composition was used to pretreat the skin histocultureas described in this Example. The skin histoculture was then treatedwith the chemotherapeutic drugs and p21/liposome treatment continued asdescribed herein. The pretreatment with p21/liposomes was found toprevent almost completely the chemotherapy induced alopecia in the skinhistoculture when compared to the control skin histoculture which wastreated with the chemotherapeutic drugs but not with the p21 liposomecomposition.

b. In Vivo Model of Chemotherapy-Induced Alopecia

8-day old Sprague-Dawley rats are treated topically with liposomecompositions as described in Example 1 containing a compound such as p21to be screened for its effect on chemotherapy-induced alopecia. Theliposome composition is provided at a concentration of 100 μg/ml in 1 mMβ-mercaptoethanol, for 2 days. In a control group, the rats do notreceive liposome pretreatment. The rats are then treated withcyclophosphamide, CTX (35 mg/kg, ip once) with the combination of CTX(25 mg/kg)+doxorubicin, DOX (2.5 mg/kg), ip once per day for 3 days,respectively. One rat pup without chemotherapy was used as control.Alopecia was noted at day-7 after chemotherapy.

In rats that received chemotherapy, alopecia can be seen in both theCTX-treated and CTX+DOX-treated rat pups. No other toxic effects werenoted.

CTX and doxorubicin can induce alopecia in rat pups by day-7 withoutother noticeable toxic effects. Rats that received the liposomecomposition pretreatment may be compared to the control rats todetermine whether the screened compound had an effect onchemotherapy-induced alopecia.

Although the present invention has now been described in terms ofcertain preferred embodiments, and exemplified with respect thereto, oneskilled in the art will readily appreciate that various modifications,changes, omissions and substitutions may be made without departing fromthe spirit thereof.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned as well as those inherent therein. The liposomecompositions, along with the methods, procedures and treatment describedherein are presently representative of preferred embodiments that areexemplary and not intended as limitations on the scope of the invention.Changes therein and other uses will occur to those skilled in the artwhich are encompassed within the spirit of the invention or defined bythis scope with the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein within departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

What is claimed is:
 1. A method to introduce a composition whichcontains at least one active ingredient which is effective to inhibitthe loss of hair caused by a chemotherapeutic agent selectively to thehair follicles of a subject without transmission to the cells externalto the hair follicles so as to prevent hair loss induced by chemotherapyin said subject in need of such prevention which methodcomprisesapplying to the skin of said subject in an area whereprevention of hair loss is desired and which contains hair follicles, aformulation comprising said composition wherein said at least one activeingredient, when contained in the cells of a hair follicle, prevents thechemotherapeutic agent-induced loss of hair extruded from said hairfollicle, said formulation being a liposomal formulation wherein saidliposomes target said composition selectively into the hair folliclecells by transfer into the follicle without entry into the circulationor the adjacent skin tissue and wherein said active ingredient is aprotein which is a cell cycle inhibitor, or wherein said activeingredient is the protein product of the multidrug resistance (MDR)gene, or wherein said composition comprises an expression system for theproduction of an effective amount of a protein which is a cell cycleinhibitor, or wherein said composition comprises an expression systemfor the production of an effective amount of the product of the MDRgene.
 2. The method of claim 1 wherein said active ingredient is aprotein which is a cell cycle inhibitor.
 3. The method of claim 1wherein said composition comprises an expression system for theproduction of an effective amount of a protein which is a cell cycleinhibitor.
 4. The method of claim 1 wherein said active ingredient isthe protein product of the multidrug resistance (MDR) gene.
 5. Themethod of claim 1 wherein said composition comprises an expressionsystem for the production of an effective amount of the product of theMDR gene.
 6. The method of claim 1 wherein the liposomes are comprisedof phosphatidylcholine (PC), phosphatidylethanolamine (PE) andcholesterol.
 7. The method of claim 1 wherein the liposomes are formedfrom egg phosphatidylcholine (EPC) or from dipalmitoylphosphatidylcholine (DPPC) or mixtures thereof.
 8. The method of claim 2wherein said cell cycle inhibitor is selected from the group consistingof p16, p15, p21, p27 and p28.
 9. The method of claim 4 where theproduct of the MDR gene is human p-glycoprotein.
 10. A liposomalformulation for delivery of a composition to hair follicles by transferinto the follicle without entry into the circulation or skin tissueadjacent to the follicle wherein said composition comprises at least oneactive ingredient which, when contained in the cells of a hair follicle,prevents chemotherapeutic agent-induced loss of hair extruded from saidhair follicle andwherein said active ingredient is a protein which is acell cycle inhibitor, or wherein said active ingredient is the proteinproduct of the multidrug resistance (MDR) gene, or wherein saidcomposition comprises an expression system for the production of aneffective amount of a protein which is a cell cycle inhibitor, orwherein said composition comprises an expression system for theproduction of an effective amount of the product of the MDR gene. 11.The formulation of claim 10 wherein the liposomes are comprised ofphosphatidylcholine (PC), phosphatidylethanolamine (PE) and cholesterol.12. The formulation of claim 10 wherein the liposomes are formed fromegg phosphatidylcholine (EPC) or from dipalmitoyl phosphatidylcholine(DPPC) or mixtures thereof.
 13. The formulation of claim 10 wherein saidactive ingredient is a protein which is a cell cycle inhibitor.
 14. Theformulation of claim 10 wherein said active ingredient is an expressionsystem for production of an effective amount of a protein which is acell cycle inhibitor.
 15. The formulation of claim 10 wherein saidactive ingredient is a protein product of the multidrug resistance (MDR)gene.
 16. The formulation of claim 10 wherein the active ingredient isan expression system for the production of an effective amount of theproduct of the MDR gene.
 17. The liposomal formulation of claim 10wherein said liposomal formulation comprisesphosphatidylcholine:phosphatidylethanolamine:cholesterol in a ratio of5:2:3.
 18. The liposomal formulation of claim 10 wherein said liposomalformulation further comprises one or more cationic phospholipidsselected from the group consisting of D282, D378, D383, D3886, D3897 andD3899.
 19. The formulation of claim 13 wherein said cell cycle inhibitoris selected from the group consisting of p16, p15, p21, p27 and p28. 20.The formulation of claim 15 wherein said protein product of the MDR geneis human p-glycoprotein.
 21. The liposomal formulation of claim 18wherein said liposomal formulation contains a molar ratio of saidphosphlipids to said cationic phospholipids of 1.0-1.2:0.8.